RESUMEN

Cystathionine gamma-synthase (CGS) catalyses the first committed step of methionine biosynthesis in higher plants. CGS is encoded by the CGS1 gene in Arabidopsis. Stability of CGS1 mRNA is down-regulated in response to methionine application and the exon 1-coding region of CGS1 itself is necessary and sufficient for this regulation. mto1 (for methionine overaccumulation) mutants of Arabidopsis, which carry single-amino-acid sequence alterations within CGS1 exon 1, are deficient in this regulation and overaccumulate methionine. Since CGS1 exon 1 acts in cis during this regulation, we have proposed a model that the regulation occurs during translation of CGS1 mRNA when the nascent polypeptide of CGS and its mRNA are in close proximity. In fact, application of the translation inhibitor cycloheximide abolished this regulation in vivo. This model predicts that the regulation can be reproduced in an in vitro translation system. Studies using the in vitro translation system of wheatgerm extract have indicated that S-adenosylmethionine, a direct metabolite of methionine, is the effector of this regulation. A 5'-truncated RNA species, which is a probable degradation intermediate of CGS1 mRNA in vivo, was also detected in vitro, suggesting that the wheatgerm in vitro translation system reflects the in vivo regulation.

Asunto(s)

Arabidopsis/enzimología , Liasas de Carbono-Oxígeno/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , S-Adenosilmetionina/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular

RESUMEN

The surfaces of land plants are covered with a cuticle that is essential for retention of water. Epidermal surfaces of Arabidopsis thaliana embryos and juvenile plants that were homozygous for abnormal leaf shape1 (ale1) mutations were defective, resulting in excessive water loss and organ fusion in young plants. In ale1 embryos, the cuticle was rudimentary and remnants of the endosperm remained attached to developing embryos. Juvenile plants had a similar abnormal cuticle. The ALE1 gene was isolated using a transposon-tagged allele ale1-1. The predicted ALE1 amino acid sequence was homologous to those of subtilisin-like serine proteases. The ALE1 gene was found to be expressed within certain endosperm cells adjacent to the embryo and within the young embryo. Expression was not detected after germination. Our results suggest that the putative protease ALE1 affects the formation of cuticle on embryos and juvenile plants and that an appropriate cuticle is required for separation of the endosperm from the embryo and for prevention of organ fusion.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/embriología , Arabidopsis/enzimología , Serina Endopeptidasas/metabolismo , Subtilisinas/metabolismo , Alelos , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Secuencia de Bases , Huella de ADN , ADN Complementario/genética , ADN de Plantas/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 , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Subtilisinas/genética

RESUMEN

Expression of the gene for cystathionine gamma-synthase (CGS), which catalyzes the key step of methionine biosynthesis, is feedback regulated at the level of mRNA stability. The first exon polypeptide of CGS is suggested to be involved in this regulation and amino acid sequence alterations caused by mto1 mutations in that region lead to an overaccumulation of CGS mRNA [Chiba et al. (1999) Science 286: 1371-1374]. Transgenic Arabidopsis thaliana harboring chimeric constructs in which wild-type or mto1 mutant CGS exon 1 are fused in-frame to reporter genes and driven by the cauliflower mosaic virus 35S RNA promoter were constructed. Studies with these transgenic lines demonstrated that the coding region of CGS exon 1 is necessary and sufficient for downregulation of its own mRNA accumulation in response to methionine application and that this region acts in cis in this process.

Asunto(s)

Arabidopsis/metabolismo , Liasas de Carbono-Oxígeno/genética , Regulación hacia Abajo , Exones , ARN Mensajero/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Secuencia de Bases , ADN Complementario , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , ARN Mensajero/genética

RESUMEN

We investigated physical distances and directions of transposition of the maize transposable element Ac in tobacco cultured cells. We introduced a T-DNA construct that carried a non-autonomous derivative of Ac (designated dAc-I-RS) that included sites for cleavage by restriction endonuclease MluI. Another cleavage site was also introduced into the T-DNA region outside of the dAc-I-RS transposable element. The tobacco cultured cell line BY-2 was transformed with the T-DNA and several transformed lines that had a single copy of the T-DNA at a different chromosomal location were isolated. These lines were co-cultured with Agrobacterium tumefaciens cells that carried a cDNA for the Ac transposase gene under the control of various promoters. Sublines of cultured cells in which dAc-I-RS had been transposed, were isolated. The genomic DNAs of these sublines were isolated and digested with MluI. Sizes of DNA segments generated by digestion were determined by pulse-field gel electrophoresis. Our results showed that 20 to 70% of transposition events had occurred within several hundreds kilo-base pairs (kb) on the same chromosome. These results demonstrate that the Ac-Ds element preferentially transposed to regions near the original site in a tobacco chromosome. In addition, the present results are an example of asymmetric transposition as demonstrated by the distance of transposition on the chromosome.

Asunto(s)

Elementos Transponibles de ADN , Nicotiana/genética , Plantas Tóxicas , Agrobacterium tumefaciens/genética , Línea Celular Transformada , Células Cultivadas , Técnicas de Cocultivo , Genes de Plantas , Modelos Genéticos , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Recombinación Genética , Zea mays/genética

RESUMEN

Flowering is often triggered by exposing plants to appropriate day lengths. This response requires an endogenous timer called the circadian clock to measure the duration of the day or night. This timer also controls daily rhythms in gene expression and behavioural patterns such as leaf movements. Several Arabidopsis mutations affect both circadian processes and flowering time; but how the effect of these mutations on the circadian clock is related to their influence on flowering remains unknown. Here we show that expression of CONSTANS (CO), a gene that accelerates flowering in response to long days, is modulated by the circadian clock and day length. Expression of a CO target gene, called FLOWERING LOCUS T (FT), is restricted to a similar time of day as expression of CO. Three mutations that affect circadian rhythms and flowering time alter CO and FT expression in ways that are consistent with their effects on flowering. In addition, the late flowering phenotype of such mutants is corrected by overexpressing CO. Thus, CO acts between the circadian clock and the control of flowering, suggesting mechanisms by which day length regulates flowering time.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/crecimiento & desarrollo , Proteínas de Unión al ADN/fisiología , Proteínas de Plantas/fisiología , Factores de Transcripción/fisiología , Arabidopsis/genética , Ritmo Circadiano , Proteínas de Unión al ADN/genética , Genes de Plantas , Mutación , Fotoperiodo , Estructuras de las Plantas/fisiología , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Factores de Transcripción/genética

RESUMEN

The vegetative growth of Arabidopsis thaliana can be divided into two phases. The transition from the juvenile (early) phase to the adult (later) phase is associated with changes in several morphological features of leaves, such as the shape of leaf blades, the number of trichomes and patterns of venation. In a screening of mutants with altered morphological identities of leaves, we found one which we named juvenile leafless and misshapen shoot apical meristem (jam). The mutation represented a new allele of the WUSCHEL (WUS) gene, and, in its presence, plants produced no juvenile leaves. Analysis of the morphology of mutant plants revealed that all the rosette leaves had characteristics of adult leaves. The formation of the first rosette leaf in the wus(jam) mutant was markedly delayed, and occurred at the almost same time as formation of the third or fourth leaf in wild-type plants. In the wild-type, these leaves correspond to the first adult leaves. Analysis by RT-PCR showed that transcripts of WUS accumulated in shoot apices and roots, but not in cotyledons and leaves. The present results suggest that the WUS gene controls the morphological traits of rosette leaves either directly or indirectly. In view of the predicted function of the WUS gene, namely maintenance of stem cells within the shoot apical meristem, we suggest that the lack of juvenile leaves in the mutant might have been caused by interruption of leaf initiation during the juvenile phase or by halting of an entire process of formation of juvenile leaves.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas de Homeodominio/genética , Arabidopsis/crecimiento & desarrollo , Cruzamientos Genéticos , ADN Bacteriano/genética , Heterocigoto , Meristema/fisiología , Mutagénesis , Fenotipo , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Brotes de la Planta/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

In plants, flowering is triggered by endogenous and environmental signals. CONSTANS (CO) promotes flowering of Arabidopsis in response to day length. Four early target genes of CO were identified using a steroid-inducible version of the protein. Two of these genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and FLOWERING LOCUS T (FT), are required for CO to promote flowering; the others are involved in proline or ethylene biosynthesis. The SOC1 and FT genes are also regulated by a second flowering-time pathway that acts independently of CO. Thus, early target genes of CO define common components of distinct flowering-time pathways.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/fisiología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica de las Plantas , Transducción de Señal , Factores de Transcripción/fisiología , Arabidopsis/genética , Cicloheximida/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Etilenos/biosíntesis , Genes de Plantas , Proteínas de Dominio MADS , Meristema/genética , Meristema/fisiología , Fenotipo , Fotoperiodo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Brotes de la Planta/genética , Brotes de la Planta/fisiología , Plantas Modificadas Genéticamente , Prolina/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta/genética , ARN de Planta/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Proteínas Recombinantes de Fusión , Supresión Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

CONSTANS (CO) promotes flowering of Arabidopsis in response to long photoperiods. Transgenic plants carrying CO fused with the cauliflower mosaic virus 35S promoter (35S::CO) flowered earlier than did the wild type and were almost completely insensitive to length of day. Genes required for CO to promote flowering were identified by screening for mutations that suppress the effect of 35S::CO. Four mutations were identified that partially suppressed the early-flowering phenotype caused by 35S::CO. One of these mutations, suppressor of overexpression of CO 1 (soc1), defines a new locus, demonstrating that the mutagenesis approach is effective in identifying novel flowering-time mutations. The other three suppressor mutations are allelic with previously described mutations that cause late flowering. Two of them are alleles of ft, indicating that FT is required for CO to promote early flowering and most likely acts after CO in the hierarchy of flowering-time genes. The fourth suppressor mutation is an allele of fwa, and fwa soc1 35S::CO plants flowered at approximately the same time as co mutants, suggesting that a combination of fwa and soc1 abolishes the promotion of flowering by CO. Besides delaying flowering, fwa acted synergistically with 35S::CO to repress floral development after bolting. The latter phenotype was not shown by any of the progenitors and was most probably caused by a reduction in the function of LEAFY. These genetic interactions suggest models for how CO, FWA, FT, and SOC1 interact during the transition to flowering.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas de Unión al ADN/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Arabidopsis/crecimiento & desarrollo , Ritmo Circadiano , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS , Mutagénesis Sitio-Dirigida , Fotoperiodo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Supresión Genética , Factores de Transcripción/metabolismo

RESUMEN

Anti-centromere autoantibodies (ACA) are commonly found in the serum of patients with a limited type of scleroderma and other systemic autoimmune diseases. CENP-A is one of the major antigens against ACA and a histone H3-like protein. To analyse the autoantigenic epitopes of CENP-A, a series of truncated peptides of human CENP-A were expressed in Escherichia coli and immunoblotting analysis was performed with 91 ACA+ sera. Eighty sera (88%) with the ACA reacted to the 52-amino acids N-terminal region which is not homologous to H3, while no sera reacted to the C-terminus which has a sequence similarity with H3. Moreover, ELISA was also employed in this study using two synthetic peptides corresponding to the amino acid sequences 3-17 (peptide A) and 25-38 (peptide B). Peptides A and B were reactive to 78 (86%) and 79 (87%) of ACA, respectively. Core antigens of hepatitis B virus (HBV) and hepatitis C virus (HCV) have similar sequences to peptide A and/or peptide B, but three sera containing HBV without ACA and five sera containing HCV without ACA were found to be reactive to neither peptide. Centromere localization of CENP-A is dependent on the H3-like C-terminal domain which is not autoantigenic, while the antigenic N-terminal domain, which might play unidentified functional roles, should be an important region for the induction of ACA.

Asunto(s)

Autoantígenos/química , Proteínas Cromosómicas no Histona/química , Epítopos/química , Histonas/química , Fragmentos de Péptidos/química , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Autoanticuerpos/sangre , Autoanticuerpos/metabolismo , Centrómero/química , Centrómero/inmunología , Centrómero/metabolismo , Proteína A Centromérica , Proteínas Cromosómicas no Histona/metabolismo , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo

RESUMEN

Flowering of Arabidopsis is promoted by long days and delayed by short days. Mutations in the GIGANTEA (GI) gene delay flowering under long days but have little or no effect under short days. We have now isolated the GI gene and show that it encodes a novel, putative membrane protein. By comparing the sequence of the Arabidopsis gene with that of a likely rice orthologue and by sequencing mutant alleles, we identify regions of the GI protein that are likely to be important for its function. We show that GI expression is regulated by the circadian clock with a peak in transcript levels 8-10 h after dawn. The timing, height and duration of this peak are influenced by daylength. We analysed the interactions between GI and the LHY, CCA1 and ELF3 genes, previously shown to affect daylength responses; we show that the rhythmic pattern of GI expression is altered in the elf3, CCA1-OX and lhy genotypes, and that CCA1 and LHY expression are reduced by gi mutations. Our results are consistent with the idea that GI plays an important role in regulating the expression of flowering time genes during the promotion of flowering by photoperiod.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/genética , Ritmo Circadiano/genética , Proteínas de Plantas/genética , Esfingomielina Fosfodiesterasa , Alelos , Secuencia de Aminoácidos , Northern Blotting , ADN Complementario/análisis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Proteínas de Plantas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transgenes

RESUMEN

We examined the clinical features of patients presenting antinuclear autoantibodies against p80-coilin and the IgG subclass distribution of anti- p80-coilin antibodies. Sera from 365 Japanese patients were analysed. Immunoblotting and indirect immunofluorescence microscopy techniques were used with a polyclonal rabbit antiserum against p80-coilin. Eleven patients with anti-p80-coilin antibodies were found. All the patients were female and nine were in their twenties. None could be diagnosed with differentiated rheumatic disease except for one case of systemic scleroderma and another of Sjögren's syndrome. Most patients had general fatigue, arthralgia, headaches, dysmenorrhea, lymph node swelling and/or low grade fever such as chronic fatigue syndrome (CFS), and showed low complement. One patient fulfilled the criteria for CFS. All were younger females than those often diagnosed with rheumatic disease in previous reports. Patients' sera had a predominant distribution of subclass IgG(1)anti-p80-coilin antibodies and five sera had concomitant subclass IgG(2). Two rheumatic disease patients had a relatively high titer of IgG(2)anti-p80-coilin antibodies. The IgG(2)subclass of anti-p80-coilin antibodies may be a specific marker for systemic autoimmune disease.

Asunto(s)

Autoanticuerpos/clasificación , Autoantígenos/inmunología , Enfermedades Autoinmunes/diagnóstico , Inmunoglobulina G/inmunología , Proteínas Nucleares/inmunología , Adulto , Núcleo Celular/ultraestructura , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Factores Sexuales

RESUMEN

Lipid delays gastric emptying, and aging is associated with changes in gastric motor function and transit. However, little is known about the effect of lipid on gastric emptying time in the elderly. To determine the effect of aging on lipid gastric emptying, we used electrical impedance tomography (EIT) to study gastric emptying of liquid meals with or without lipid in five young (23.0 +/- 0.6 years, mean +/- SEM) and six elderly (73.3 +/- 1.6 years) healthy male volunteers. These subjects drank 400 ml of non-lipid soup (triglycerides, 0 g) or lipid soup (triglycerides, 24.6g) in liquid test meals. To study the effect of lipolysis in the stomach, a liquid test meal containing 240mg of lipase in the lipid soup was also administered. Plasma cholecystokinin (CCK) concentration was measured by specific radioimmunoassay before and 30 min after the ingestion of a test meal. The gastric emptying time of the lipid soup was longer in the elderly than in the young subjects, and the time was significantly longer for lipid soup than for nonlipid soup (P < 0.05) in both the young and elderly subjects. Gastric emptying time for non-lipid soup was not significantly different between the elderly and young subjects. The administration of lipase shortened the gastric emptying time for lipid in both the elderly and the young subjects. Basal CCK concentration was significantly higher in the elderly than in the young subjects. However, there was no relationship between gastric emptying time and plasma CCK concentration after the ingestion of a test meal in the subjects overall. In conclusion, the delaying effect of lipid on gastric emptying is increased in the elderly, and the administration of lipase accelerates the emptying of lipid from the stomach.

Asunto(s)

Envejecimiento/fisiología , Vaciamiento Gástrico/fisiología , Lípidos/fisiología , Lipólisis/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Colecistoquinina/sangre , Impedancia Eléctrica , Vaciamiento Gástrico/efectos de los fármacos , Humanos , Lipasa/farmacología , Metabolismo de los Lípidos , Lipólisis/efectos de los fármacos , Masculino , Factores de Tiempo , Tomografía

RESUMEN

An NADPH oxidase analogous to that in mammalian phagocytes has been hypothesized to produce reactive oxygen species (ROS) in the plant defence response. A. thaliana contains at least six gp91phox homologues, designated AtrbohA-F (A. thaliana Respiratory Burst Oxidase Homologues), which map to different positions. Transcripts of three of these genes can be detected in healthy plants by RNA gel blot analyses. The Atrboh gene products are closely related to gp91phox and the intron locations suggest a common evolutionary origin. A putative EF-hand Ca(2+)-binding motif in the extended N-terminal region of the Atrboh proteins suggests a direct regulatory effect of Ca2+ on the activity of the NADPH oxidase in plants.

Asunto(s)

Arabidopsis/genética , Glicoproteínas de Membrana/genética , NADPH Oxidasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Humanos , Datos de Secuencia Molecular , NADPH Oxidasa 2 , Estallido Respiratorio , Homología de Secuencia de Aminoácido

RESUMEN

We have investigated physical distances and directions of transposition of the maize transposable element Ac in Arabidopsis thaliana. We prepared a transferred DNA (T-DNA) construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. Three transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. These transgenic plants were crossed with the Arabidopsis that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with endonuclease I-SceI, sizes of segment of DNA were determined by pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results showed that 50% of all transposition events had occurred within 1,700 kb on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac-Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites.

Asunto(s)

Autoanticuerpos/inmunología , Neumatosis Cistoide Intestinal/inmunología , Antígeno Nuclear de Célula en Proliferación/inmunología , Adulto , Enfermedad Crónica , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Seudoobstrucción Intestinal/inmunología , Seudoobstrucción Intestinal/patología , Neumatosis Cistoide Intestinal/patología

RESUMEN

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic beta-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.

Asunto(s)

Arabidopsis/genética , ADN Nucleotidiltransferasas/metabolismo , Proteínas Fúngicas/metabolismo , Recombinación Genética , Saccharomycetales/enzimología , Arabidopsis/metabolismo , Secuencia de Bases , Cruzamientos Genéticos , ADN Nucleotidiltransferasas/genética , Cartilla de ADN , Proteínas Fúngicas/genética , Genes Reporteros , Germinación , Glucuronidasa/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plantas Modificadas Genéticamente , Saccharomycetales/genética

RESUMEN

Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.

Asunto(s)

ADN Nucleotidiltransferasas/genética , ADN Recombinante , Integrasas , Nicotiana/genética , Plantas Tóxicas , Saccharomycetales/genética , Secuencia de Bases , Células Cultivadas , Clonación Molecular , ADN Nucleotidiltransferasas/metabolismo , ADN de Hongos , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Recombinasas , Saccharomycetales/enzimología , Transfección