RESUMEN
Female fertility plays a decisive role in the reproduction of mammals, with related issues that include oocyte or embryo quality, establishment of pregnancy, and the physiology of the tissues that contribute to reproduction and metabolic disorders associated with reproductive failure. Although reproductive failure may be attributed to various factors in different species, female infertility is largely controlled by a number of molecular signals that can be regulated in a cycle- and tissue-dependent manner.
Asunto(s)
Líquido Folicular/química , MicroARNs/análisis , Técnicas Reproductivas Asistidas , Femenino , Humanos , Infertilidad Femenina/terapia , Oocitos , EmbarazoRESUMEN
During the last decade, a large number of QTLs and candidate genes for rice tolerance to salinity have been reported. Using 124 SNP and 52 SSR markers, we targeted 14 QTLs and 65 candidate genes for association mapping within the European Rice Core collection (ERCC) comprising 180 japonica accessions. Significant differences in phenotypic response to salinity were observed. Nineteen distinct loci significantly associated with one or more phenotypic response traits were detected. Linkage disequilibrium between these loci was extremely low, indicating a random distribution of favourable alleles in the ERCC. Analysis of the function of these loci indicated that all major tolerance mechanisms were present in the ERCC although the useful level of expression of the different mechanisms was scattered among different accessions. Under moderate salinity stress some accessions achieved the same level of control of Na(+) concentration and Na(+)/K(+) equilibrium as the indica reference variety for salinity tolerance Nona Bokra, although without sharing the same alleles at several loci associated with Na(+) concentration. This suggests (a) differences between indica and japonica subspecies in the effect of QTLs and genes involved in salinity tolerance and (b) further potential for the improvement of tolerance to salinity above the tolerance level of Nona Bokra, provided the underlying mechanisms are complementary at the whole plant level. No accession carried all favourable alleles, or showed the best phenotypic responses for all traits measured. At least nine accessions were needed to assemble the favourable alleles and all the best phenotypic responses. An effective strategy for the accumulation of the favourable alleles would be marker-assisted population improvement.
Asunto(s)
Homeostasis , Oryza/genética , Potasio/metabolismo , Plantas Tolerantes a la Sal/genética , Sodio/metabolismo , Alelos , Estudios de Asociación Genética , Marcadores Genéticos , Variación Genética , Genotipo , Desequilibrio de Ligamiento , Oryza/fisiología , Presión Osmótica , Fenotipo , Sitios de Carácter Cuantitativo , Salinidad , Plantas Tolerantes a la Sal/fisiología , Cloruro de SodioRESUMEN
Insertional mutant databases containing Flanking Sequence Tags (FSTs) are becoming key resources for plant functional genomics. We have developed OryGenesDB (http://orygenesdb.cirad.fr/), a database dedicated to rice reverse genetics. Insertion mutants of rice genes are catalogued by Flanking Sequence Tag (FST) information that can be readily accessed by this database. Our database presently contains 44166 FSTs generated by most of the rice insertional mutagenesis projects. The OryGenesDB genome browser is based on the powerful Generic Genome Browser (GGB) developed in the framework of the Generic Model Organism Project (GMOD). The main interface of our web site displays search and analysis interfaces to look for insertions in any candidate gene of interest. Several starting points can be used to exhaustively retrieve the insertions positions and associated genomic information using blast, keywords or gene name search. The toolbox integrated in our database also includes an 'anchoring' option that allows immediate mapping and visualization of up to 50 nucleic acid sequences in the rice Genome Browser of OryGenesDB. As a first step toward plant comparative genomics, we have linked the rice and Arabidopsis whole genome using all the predicted pairs of orthologs by best BLAST mutual hit (BBMH) connectors.
Asunto(s)
Bases de Datos Genéticas , Genes de Plantas , Mutagénesis Insercional , Oryza/genética , Mapeo Cromosómico , Genoma de Planta , Genómica , Internet , Proteínas de Plantas/genética , Lugares Marcados de Secuencia , Interfaz Usuario-ComputadorRESUMEN
The first bacterial artificial chromosome (BAC) library of Robusta coffee (Coffea canephora) was constructed, with the aim of developing molecular resources to study the genome structure and evolution of this perennial crop. Clone 126, which is highly productive and confers good technological and organoleptic qualities of beverage, was chosen for development of this library. The BAC library contains 55,296 clones, with an average insert size of 135 Kb per plasmid, therefore representing theoretically nine haploid genome equivalents of C. canephora. Its validation was achieved with a set of 13 genetically anchored single-copy and 4 duplicated RFLP probes and yielded on average 9 BAC clones per probe. Screening of this BAC library was also carried out with partial cDNA probes coding for enzymes of sugar metabolism like invertases and sucrose synthase, with the aim of characterizing the organization and promoter structure of this important class of genes. It was shown that genes for both cell wall and vacuolar forms of invertases were probably unique in the Robusta genome whereas sucrose synthase was encoded by at least two genes. One of them (CcSUS1) was cloned and sequenced, showing that our BAC library is a valuable tool to rapidly identify genes of agronomic interest or linked to cup quality in C. canephora.
Asunto(s)
Mapeo Cromosómico , Cromosomas Artificiales Bacterianos , Coffea/genética , Biblioteca de Genes , Glucosiltransferasas/genética , beta-Fructofuranosidasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , Cartilla de ADN , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
We have constructed and validated the first cocoa ( Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp ( palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.
Asunto(s)
Cacao/genética , Cacao/fisiología , Cromosomas Artificiales Bacterianos/genética , Genómica/métodos , Mapeo Físico de Cromosoma/métodos , Enfermedades de las Plantas/genética , Cacao/parasitología , Cromosomas de las Plantas/genética , Mapeo Contig , Biblioteca de Genes , Marcadores Genéticos/genética , Genoma de Planta , Genotipo , Fenotipo , Phytophthora/fisiología , Enfermedades de las Plantas/parasitología , Reproducibilidad de los ResultadosRESUMEN
A bacterial artificial chromosome (BAC) library for banana was constructed from leaves of the wild diploid 'Calcutta 4' clone (Musa acuminata subsp. Burmannicoides 2n = 2 x = 22). 'Calcutta 4' is widely used in breeding programs for its resistance to the current major disease of banana and is being used to build a genetic reference map of banana. As banana leaves are particularly rich in polyphenols and polysaccharides a protocol was adapted to isolate intact nuclei and high-molecular-weight (HMW) DNA. A total of 55,152 clones with an average insert size of 100 kb were picked. The frequency of BAC clones carrying inserts derived from chloroplast and mitochondrial DNA was estimated to be 1.5%. The coverage of the library is equivalent to 9.0-times the haploid genome. The BAC library was screened with 13 RFLP probes belonging to the 8 linkage groups of the consensus molecular map of banana. A total of 135 clones were identified giving an average of 10.38 clones for each locus. This BAC library will be a valuable starting tool for many of the goals of the recently emerged International Musa Genomic Consortium. One of our initial objectives will be to develop a banana physical map by BAC-FISH (fluorescent in situ hybridization) viewing the characterization of translocation break points.
Asunto(s)
Cromosomas Artificiales Bacterianos , Biblioteca de Genes , Musa/genética , ADN de Cloroplastos , ADN MitocondrialRESUMEN
Barley Mlo defines the founder of a novel class of plant integral membrane proteins. Lack of the wild type protein leads to broad spectrum disease resistance against the pathogenic powdery mildew fungus and deregulated leaf cell death. Scanning N-glycosylation mutagenesis and Mlo-Lep fusion proteins demonstrated that Mlo is membrane-anchored by 7 transmembrane (TM) helices such that the N terminus is located extracellularly and the C terminus intracellularly. Fractionation of leaf cells and immunoblotting localized the protein to the plant plasma membrane. A genome-wide search for Mlo sequence-related genes in Arabidopsis thaliana revealed approximately 35 family members, the only abundant gene family encoding 7 TM proteins in higher plants. The sequence variability of Mlo family members within a single species, their topology and subcellular localization are reminiscent of the most abundant class of metazoan 7 TM receptors, the G-protein-coupled receptors.
Asunto(s)
Hordeum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Aminoácidos/metabolismo , Animales , Arabidopsis/genética , Secuencia de Bases , Caenorhabditis elegans/genética , Membrana Celular/metabolismo , Perros , Genoma de Planta , Glicosilación , Hordeum/genética , Microsomas/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Pancrelipasa/metabolismo , Proteínas de Plantas/química , Proteínas Recombinantes de Fusión , Homología de Secuencia de Ácido NucleicoRESUMEN
The combination of mutational and molecular studies has shed light on the role of reactive oxygen intermediates and programmed cell death in cereal disease resistance mechanisms. Rice Rac1 and barley Rar1 represent conserved disease resistance signalling genes, which may have related functions in animals. The analysis of non-pathogenic Magnaporthe grisea mutants may provide novel tools to study host defence pathways.
Asunto(s)
Grano Comestible/metabolismo , Transducción de Señal , Animales , Apoptosis , Transducción de Señal/genéticaRESUMEN
The contiguous DNA sequence of a 60 kb genomic interval of barley chromosome 4HL has been assembled. The region harbours a single and novel gypsy -like retrotransposon, designated BAGY-1. Only three genes appear to reside in the genomic stretch. One predicts a plant homologue of ribophorin I, a subunit of the oligosaccharyltransferase-protein complex located in the rough endoplasmatic reticulum. The second is similar to the Drosophila g1 gene encoding a ring finger protein involved in developmental processes. The observed gene density is approximately 5-fold lower than in the best characterized dicot genome of Arabidopsis but 6- to 10-fold higher than expected from an equidistant gene distribution in the complex barley genome. Our data suggest that the 60 kb genomic interval represents part of a gene island, a seemingly distinctive feature of grass genomes.
Asunto(s)
Islas de CpG , ADN de Plantas/genética , Genoma de Planta , Hordeum/genética , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Composición de Base , Drosophila/genética , Expresión Génica , Genes de Insecto , Genes de Plantas , Humanos , Intrones , Datos de Secuencia Molecular , Retroelementos , Especificidad de la Especie , Zea mays/genéticaRESUMEN
The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.
Asunto(s)
Arabidopsis/genética , Mapeo Cromosómico , Genoma de Planta , Cromosomas Artificiales de Levadura , Genes de Plantas/fisiología , Familia de Multigenes , Proteínas de Plantas/genética , Análisis de Secuencia de ADNRESUMEN
Pollen development in angiosperms is regulated by the interaction of products contributed by both the gametophytic (haploid) and sporophytic (diploid) genomes. In entomophilous species, lipids are major products of both sporophytic and gametophytic metabolism during pollen development. Mature pollen grains of Brassica napus are shown to contain three major acyl lipid pools as follows: (i) the extracellular tryphine mainly consisting of medium-chain neutral esters; (ii) the intracellular membranes, particularly endoplasmic reticulum, mainly containing phospholipids; and (iii) the intracellular storage lipids, which are mostly triacylglycerols. This paper reports on the kinetics of accumulation of these lipid classes during pollen maturation and the expression patterns of several lipid biosynthetic genes and their protein products that are differentially regulated in developing microspores/ pollen grains (gametophyte) and tapetal cells (sporophyte) of B. napus. Detailed analysis of three members of the stearoyl-ACP desaturase (sad) gene family by Northern blotting, in situ hybridization and RT-PCR showed that the same individual genes were expressed both in gametophytic and sporophytic tissues, although under different temporal regulation. In the tapetum, maximal expression of two marker genes for lipid biosynthesis (sad and ear) occurred at a bud length of 2-3 mm, and the corresponding gene products SAD and EAR were detected by Western blotting in 3-4 mm buds, coinciding with the maximal rates of tapetal lipid accumulation. These lipids are released following tapetal cell disintegration and are relocated to form the major structural component of the extracellular tryphine layer that coats the mature pollen grain. In contrast, in developing microspores/pollen grains, maximal expression of the lipid marker genes sad, ear, acp and cyb5 was at the 3-5 mm bud stages, with the SAD and EAR gene products detected in 4-7 mm buds. This pattern of expression coincided with accumulation of the intracellular storage and membrane lipid components of pollen. These results suggest that, although the same genes may be expressed in the sporophytic tapetal cells and in gametophytic tissues, they are regulated differentially leading to the production of the various contrasting lipidic structures that are assembled together to give rise to a viable, fertile pollen grain.
Asunto(s)
Brassica/fisiología , Regulación de la Expresión Génica de las Plantas , Lípidos/biosíntesis , Oxigenasas de Función Mixta/biosíntesis , Polen/fisiología , Transcripción Genética , Cartilla de ADN , Ésteres , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Hibridación in Situ , Lípidos/aislamiento & purificación , Microscopía Electrónica , Datos de Secuencia Molecular , Polen/ultraestructura , Reacción en Cadena de la Polimerasa , Triglicéridos/biosíntesis , Triglicéridos/aislamiento & purificaciónRESUMEN
A bidirectional promoter can be defined operationally as a short segment of DNA that regulates divergent transcription. In an attempt to investigate whether the intergenic region between the oleosin and a second open reading frame (ORFII) in Brassica napus (L.) is a divergent promoter, and also to characterize the ORFII, cDNA clones homologous to ORFII were isolated from a leaf cDNA library. A representative cDNA (clone D) of one of the two classes identified was identical, in DNA sequence, to the genomic ORFII. The second representative cDNA (clone O) was 97% identical at the nucleotide level to the genomic ORFII. The predicted amino acid sequence of the cDNA clones each exhibit homology with the peptide methionine sulphoxide reductase (PMSR) of Escherichia coli. The gene structure of ORFII was elucidated and the relative positions of the oleosin, ORFII, and the intergenic promoter region were determined. This confirms that the B. napus oleosin-ORFII intergenic region has divergent promoter activity. Consequently this is the first such plant nuclear divergent promoter identified. RFLP-mapping results showed that all four ORFII genes are linked to four of the six copies of the oleosin genes. This suggested that the bidirectional promoter locus is conserved within the B. napus genome. The ORFII gene product is targeted to the chloroplast, which is consistent with previous data indicating the presence of PMSR activity in the chloroplast. The over-expressed recombinant fusion protein (minus the transitpeptide) showed the capability to reduce peptide methionine sulphoxide residues in vitro, indicating PMSR activity. This study demonstrates that ORFII is transcribed and encodes a plant PMSR, and is the first example of the isolation of a eukaryotic PMSR gene.
Asunto(s)
Brassica/enzimología , Brassica/genética , Proteínas de Unión al ADN/genética , Genes de Plantas , Oxidorreductasas/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cloroplastos/enzimología , Cloroplastos/genética , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/genética , Ligamiento Genético , Metionina Sulfóxido Reductasas , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/enzimologíaRESUMEN
The nucleotide sequence of a Brassica napus stearoyl-acyl carrier protein desaturase gene (Bn10) is presented. This gene is one member of a family of four closely related genes expressed in oilseed rape. The expression of the promoter of this gene in transgenic tobacco was found to be temporally regulated in the developing seed tissues. However, the promoter was also particularly active in other oleogenic tissues such as the tapetum and pollen grains. This raises the interesting question of whether seed-expressed lipid synthesis genes are regulated by separate tissue-specific determinants or by a single factor common to all oleogenic tissues. Parts of the plants undergoing rapid development such as the components of immature flowers and seedlings also exhibited high levels of promoter activity. These tissues are likely to have an elevated requirement for membrane lipid synthesis. Stearoyl-acyl carrier protein desaturase transcript levels have previously been shown to be temporally regulated in the B. napus embryo (S.P. Slocombe, I. Cummins, R.P. Jarvis, D.J. Murphy [1992] Plant Mol Biol 20: 151-155). Evidence is presented demonstrating the induction of desaturase mRNA by abscisic acid in the embryo.
Asunto(s)
Brassica/enzimología , Brassica/genética , Regulación Enzimológica de la Expresión Génica , Genes de Plantas , Oxigenasas de Función Mixta/biosíntesis , Oxigenasas de Función Mixta/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Exones , Glucuronidasa/biosíntesis , Glucuronidasa/metabolismo , Cinética , Datos de Secuencia Molecular , Plantas Tóxicas , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Mapeo Restrictivo , Rhizobium/genética , Homología de Secuencia de Ácido Nucleico , Factores de Tiempo , Nicotiana/genética , TransfecciónRESUMEN
In Brassica napus, oleosins are expressed at high levels in the seed during the latter stages of embryo development. The cis-acting regulatory properties of an 872 bp promoter fragment of a B. napus oleosin gene were examined by analysis of beta-glucuronidase (GUS) expression in transgenic tobacco plants containing an oleosin promoter-GUS transcriptional fusion. The reporter gene was expressed at high levels only in seeds, specifically in embryo and endosperm tissue and regulated throughout seed development. These data demonstrate that oleosin gene transcription is regulated in a tissue-specific and temporally regulated manner and clearly indicate that oleosin protein expression is co-ordinated primarily at the transcriptional level. Oleosin mRNA was shown to be abscisic acid (ABA) inducible and an ABA-response element in the oleosin promoter was shown to be bound by a protein factor in a sequence-specific manner. Sequence analysis of the oleosin promoter has identified several other putative cis-acting sequences which may direct oleosin gene expression. The presence of a large open reading frame in the bottom strand of the oleosin promoter (ORF2) which encodes a polypeptide similar to the ethylene-induced E4 gene of tomato is reported. A PCR-generated DNA probe containing the ORF2 sequence hybridised with a 1.4 kb transcript in total RNA extracts of a variety of tissues, including leaves and germinated seed cotyledons. This finding suggests that the oleosin gene promoter directs transcription in both directions. It is the first report of a bi-directional nuclear gene promoter in plants.
Asunto(s)
Brassica/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Semillas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Guanosina/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Secuencias Reguladoras de Ácidos Nucleicos , Homología de Secuencia de AminoácidoRESUMEN
Molecular cloning of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota was achieved by immunoscreening of a cDNA library obtaining a 2 kbp clone which contains an open reading frame of 1528 bp. Comparison of the deduced amino acid sequence with those from other sources revealed the presence of motifs typical of DHFR and TS thus confirming the bifunctional nature of the carrot protein. As in other organisms, a higher degree of conservation was observed in the TS domain. Analysis of the dhfr-ts gene content in carrot revealed the presence of several copies per diploid genome.
Asunto(s)
Complejos Multienzimáticos/genética , Plantas/enzimología , Plantas/genética , Tetrahidrofolato Deshidrogenasa/genética , Timidilato Sintasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Leishmania tropica/enzimología , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
The possibility that chromosome alterations take place in personnel exposed to electromagnetic radiation in radar centres has been studied. The study was carried out "blind" on short-term cultures of peripheral blood, some 450 metaphases being analysed for each case. No aberrations were found in either radar operators or controls and there was no statistically significant difference between them.