Análise quantitativa de rna mensageiros, proteínas e atividades enzimáticas no estudo da rede de regulação controlada pelo gene Opaco-2

The Opaque-2 (O2) gene encodes a transcriptional activator specifically expressed for grain development of maize. o2 mutants have an opaque and chalky kernel, with a decrease in zein storage protein content, and an increase in the proportions of lysine and tryptophan. In this review, we present recent results investigating genetic properties of the O2 network, using transcriptome and proteome approaches, associated with measurements of activities of enzymes of the aspartate pathway and lysine degradation. The structural polymorphism at the O2 locus was investigated by RFLP in a collection of 51 maize inbred lines. Most polymorphic sites were found outside the coding regions. We then searched for relationships between RFLP polymorphism and (i) mRNA abundance of O2 and of known or suspected target genes, (ii) activity of SDH and (iii) amount of zein isoforms. Polymorphic restriction sites in the 5' upstream regions of the O2 gene were found associated with O2 mRNA abundance (three sites) and the amount of two 19 kDa alpha-zein isoforms (two sites). One restriction site on the 3' side of the O2 gene was found associated with Lor/Sdh mRNA abundance. Our results indicate relationships between polymorphism at the O2 locus and the expression of some of its target genes. Evidence of these associations has to be confirmed on larger samples, and the analysis of the O2 gene sequence should allow more precise testing of the actual involvement of O2 polymorphism in its own transcriptional expression, and in the expression of its target genes.O gene Opaco-2 (O2), expresso especificamente no grão de milho, transcreve para um fator de transcrição da família "leucine-zipper". Mutantes o2 apresentam grãos opacos, redução na quantidade de zeínas e aumento na proporção de lisina e triptofano. Genes cuja expressão é controlada diretamente pelo O2 são conhecidos (alfa-zeínas de 22 kDa, beta-zeínas de 14 kDa, b-32 e cyPpdk1). Nesta revisão, nós apresentamos resultados da caracterização genética de genes relacionados com o O2, através de abordagens de transcritoma, proteoma e de atividades enzimáticas da via metabólica do aspartato e da degradação da lisina. O polimorfismo do locus O2 foi avaliado utilisando-se a técnica de RFLP em 51 linhagens de milho. A maioria dos polimorfismos foi observada nas regiões não codificadoras da proteína. Análises de correlação foram realizadas entre os polimorfismos de RFLP e (i) quantidade de RNAm do O2, cyPpdk, Lor/Sdh e Ahas (ii) quantidade de isoformas de zeínas e (iii) atividade da enzima SDH. Sítios polimórficos foram correlacionados com a quantidade de RNAm do próprio O2, do gene Lor/Sdh e com a quantidade de duas isoformas de a-zeinas de 19 kDa. Nossos resultados indicam a presença de relações entre o polimorfismo do locus O2 e o nível de expressão de genes sob o seu controle. A utilização de um maior número de linhagens e o uso de dados de seqüência do O2 permitirá uma análise precisa da conseqüência do polimorfismo deste fator de transcrição sobre o controle do seu próprio nível de expressão e dos genes por ele controlados

Unraveling the Developmental and Genetic Mechanisms Underpinning Floral Architecture in Proteaceae

Proteaceae are a basal eudicot family with a highly conserved floral groundplan but which displays considerable variation in other aspects of floral and inflorescence morphology. Their morphological diversity and phylogenetic position make them good candidates for understanding the evolution of floral architecture, in particular the question of the homology of the undifferentiated perianth with the differentiated perianth of core eudicots, and the mechanisms underlying the repeated evolution of zygomorphy. In this paper, we combine a morphological approach to explore floral ontogenesis and a transcriptomic approach to access the genes involved in floral organ identity and development, focusing on Grevillea juniperina, a species from subfamily Grevilleoideae. We present developmental data for Grevillea juniperina and three additional species that differ in their floral symmetry using stereomicroscopy, SEM and High Resolution X-Ray Computed Tomography. We find that the adnation of stamens to tepals takes place at early developmental stages, and that the establishment of bilateral symmetry coincides with the asymmetrical growth of the single carpel. To set a framework for understanding the genetic basis of floral development in Proteaceae, we generated and annotated de novo a reference leaf/flower transcriptome from Grevillea juniperina. We found Grevillea homologs of all lineages of MADS-box genes involved in floral organ identity. Using Arabidopsis thaliana gene expression data as a reference, we found homologs of other genes involved in floral development in the transcriptome of G. juniperina. We also found at least 21 class I and class II TCP genes, a gene family involved in the regulation of growth processes, including floral symmetry. The expression patterns of a set of floral genes obtained from the transcriptome were characterized during floral development to assess their organ specificity and asymmetry of expression

Molecular and Evolutionary Bases of Within-Patient Genotypic and Phenotypic Diversity in Escherichia coli Extraintestinal Infections

Although polymicrobial infections, caused by combinations of viruses, bacteria, fungi and parasites, are being recognised with increasing frequency, little is known about the occurrence of within-species diversity in bacterial infections and the molecular and evolutionary bases of this diversity. We used multiple approaches to study the genomic and phenotypic diversity among 226 Escherichia coli isolates from deep and closed visceral infections occurring in 19 patients. We observed genomic variability among isolates from the same site within 11 patients. This diversity was of two types, as patients were infected either by several distinct E. coli clones (4 patients) or by members of a single clone that exhibit micro-heterogeneity (11 patients); both types of diversity were present in 4 patients. A surprisingly wide continuum of antibiotic resistance, outer membrane permeability, growth rate, stress resistance, red dry and rough morphotype characteristics and virulence properties were present within the isolates of single clones in 8 of the 11 patients showing genomic micro-heterogeneity. Many of the observed phenotypic differences within clones affected the trade-off between self-preservation and nutritional competence (SPANC). We showed in 3 patients that this phenotypic variability was associated with distinct levels of RpoS in co-existing isolates. Genome mutational analysis and global proteomic comparisons in isolates from a patient revealed a star-like relationship of changes amongst clonally diverging isolates. A mathematical model demonstrated that multiple genotypes with distinct RpoS levels can co-exist as a result of the SPANC trade-off. In the cases involving infection by a single clone, we present several lines of evidence to suggest diversification during the infectious process rather than an infection by multiple isolates exhibiting a micro-heterogeneity. Our results suggest that bacteria are subject to trade-offs during an infectious process and that the observed diversity resembled results obtained in experimental evolution studies. Whatever the mechanisms leading to diversity, our results have strong medical implications in terms of the need for more extensive isolate testing before deciding on antibiotic therapies

Comparaison de six espèces de luzernes annuelles à l'aide de caractères biométriques et enzymatiques

International audienc

ANALYSE DU POLYMORPHISME DU GENE OPAQUE-2 CHEZ LE MAIS (ZEA MAYS L.) ET RECHERCHE DE FACTEURS GENETIQUES IMPLIQUES DANS LA VARIATION QUANTITATIVE DE SON TRANSCRIT

LES ANALYSES GENETIQUES ET MOLECULAIRES ONT MONTRE QUE LE LOCUS OPAQUE-2 (O2) CODE POUR UN FACTEUR DE TRANSCRIPTION DE LA FAMILLE DES LEUCINE-ZIPPER, ET EST IMPLIQUE DANS LE CONTROLE DE L'EXPRESSION COORDONNEE DE PLUSIEURS GENES DES VOIES DE BIOSYNTHESE DES RESERVES DE L'ALBUMEN (PROTEIQUES OU CARBONEES). LE GENE O2 EST DE CE FAIT UN MODELE D'ETUDE ORIGINAL POUR EXAMINER COMMENT LE POLYMORPHISME AU NIVEAU D'UN FACTEUR DE REGULATION PEUT JOUER UN ROLE DANS LA VARIATION DE CARACTERES PHENOTYPIQUES. LA COMPARAISON DE SEQUENCES DU GENE O2 DE HUIT ALLELES MUTANTS ET SAUVAGES A PERMIS DE CARACTERISER LES ALTERATIONS MOLECULAIRES RESPONSABLES DU PHENOTYPE MUTANT. NOUS AVONS MIS EN EVIDENCE DEUX TYPES D'ALLELES : L'UN EST DU A UNE DELETION DANS LA REGION CODANTE QUI CONDUIT A UNE PROTEINE TRONQUEE NON FONCTIONNELLE. L'AUTRE EST UN ALLELE NUL CAR IL NE POSSEDE NI TRANSCRITS, NI PROTEINE O2. LE DEUXIEME ASPECT DU TRAVAIL A PORTE SUR DES ALLELES SAUVAGES ET LEUR ACTIVITE TRANSCRIPTIONNELLE. LA RECHERCHE D'ELEMENTS CIS ET TRANS REGULATEURS DE L'EXPRESSION DU GENE O2 A ETE REALISEE A L'AIDE DE DEUX STRATEGIES. L'ETUDE D'ASSOCIATIONS STATISTIQUES ENTRE LE POLYMORPHISME RFLP AU LOCUS O2 ET LA QUANTITE D'ARNM O2 AU SEIN D'UNE POPULATION DE 51 LIGNEES DIVERSIFIEES A PERMIS DE METTRE EN EVIDENCE L'EXISTENCE D'ELEMENTS CIS EN AMONT DU GENE, REGULANT LA QUANTITE DE TRANSCRITS DANS DES GRAINS AGES DE 20 JOURS. UNE RECHERCHE DE QTL DE LA QUANTITE D'ARNM O2 AU SEIN D'UNE POPULATION DE 121 LIGNEES RECOMBINANTES REVELE EGALEMENT L'IMPLICATION DE PLUSIEURS REGULATEURS POLYMORPHES AGISSANT EN TRANS SUR LA VARIABILITE DE LA QUANTITE DE TRANSCRITS AU STADE PRECOCE DU GRAIN. CE TRAVAIL A MONTRE L'INTERET DES DEUX APPROCHES UTILISEES POUR LA MISE EN EVIDENCE DE FACTEURS POLYMORPHES REGULANT L'EXPRESSION D'UN GENE. L'ANALYSE DU POLYMORPHISME DE LA QUANTITE DE PROTEINE O2 S'AVERERAIT UNE ETUDE COMPLEMENTAIRE TRES PERTINENTE POUR EVALUER L'IMPLICATION DU GENE O2 DANS LA REGULATION DE CES GENES CIBLES.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

ANALYSE DE LA VARIABILITE D'EXPRESSION ET DE STRUCTURE DE GENES CIBLES DU FACTEUR DE TRANSCRIPTION OPAQUE-2 CHEZ LE MAIS (ZEA MAYS L.) - RECHERCHE D'ASSOCIATIONS AVEC LE POLYMORPHISME AU LOCUS O2

LE FACTEUR DE TRANSCRIPTION OPAQUE-2 ET LES GENES SOUS SON CONTROLE REPRESENTENT UN SYSTEME DE REGULATION POTENTIELLEMENT IMPORTANT VIS-A-VIS DE LA COMPOSITION DU GRAIN DE MAIS. L'OBJET DE CETTE THESE EST L'EVALUATION DU POLYMORPHISME DE STRUCTURE ET D'EXPRESSION DE CERTAINS GENES CIBLES D'O2, ET LA RECHERCHE DE POLYMORPHISMES AU LOCUS O2 RESPONSABLES DE LA VARIABILITE OBSERVEE. NOUS AVONS EVALUE LES POLYMORPHISMES DES GENES AHAS108 ET CYPPDK1 PAR CAPS, ET LEURS QUANTITES D'ARNM AINSI QUE CELLE DU GENE LKR/SDH DANS LES GRAINS EN DEVELOPPEMENT PAR NORTHERN BLOT. LES PROTEINES DE ZEINES TOTALES, ET 42 ISOFORMES DE ZEINES REVELEES PAR ELECTROPHORESE BIDIMENSIONNELLE ONT ETE QUANTIFIEES DANS LES GRAINS MURS. UNE POPULATION DE 53 LIGNEES ET 6 MUTANTS O2 ONT ETE UTILISES. LE SEQUENCAGE D'ALLELES SAUVAGES ET MUTANTS A PERMIS D'IDENTIFIER UN ALLELE MUTANT PRODUISANT UNE PROTEINE NON FONCTIONNELLE ET UN ALLELE NUL. LA PRESENCE D'UNE PROTEINE NON FONCTIONNELLE ENGENDRE UNE REDUCTION PLUS DRASTIQUE DE LA QUANTITE DES -ZEINES DE 22 KDA ET DES -ZEINES DE 14 KDA. LE GENE CYPPDK1 S'EST REVELE PLUS POLYMORPHE DANS LES REGIONS CODANTES QUE LE GENE AHAS108, ALORS QUE LES NIVEAUX DE POLYMORPHISME SONT COMPARABLES AU NIVEAUX DES PROMOTEURS. UN EFFET GENETIQUE SIGNIFICATIF A ETE OBSERVE POUR TOUS LES CARACTERES, SAUF POUR QUELQUES ISOFORMES DE ZEINES. CERTAINES LIGNEES ONT UN PHENOTYPE PROCHE DES MUTANTS, SUGGERANT UN CONTROLE POLYGENIQUE DE L'EXPRESSION DES GENES CIBLES. LE NOMBRE REDUIT DE SITES POLYMORPHES RFLP AU LOCUS O2 ASSOCIE AUX CARACTERES RENFORCE CETTE HYPOTHESE. TOUTEFOIS, D'AUTRES FACTEURS PEUVENT EXPLIQUER CE RESULTAT : I) LES SITES RESPONSABLES DE LA VARIABILITE DES CARACTERES N'ONT PAS ETE MIS EN EVIDENCE II) LE FAIBLE EFFECTIF REND IMPOSSIBLE LA SEPARATION DE L'EFFET DE L'ALLELE DE L'EFFET DU FOND GENETIQUE. L'HYPOTHESE D'UN ROLE IMPORTANT D'O2 DANS LA VARIABILITE DES GENES ETUDIES DEVRAIT ETRE PRECISEE PAR LA RECHERCHE D'ASSOCIATIONS AVEC LES DONNEES DE SEQUENCE D'O2.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Identification of proteins regulated by cross-talk between drought and hormone pathways in Arabidopsis wild-type and auxin-insensitive mutants, axr1 and axr2

Ten proteins differentially regulated by progressive drought stress in Arabidopsis Columbia wild-type, axr1-3 and axr2-1auxin-insensitive mutants, were identified from internal amino acid microsequencing. These proteins fell into two categories: (i) stress-related proteins, known to be induced by rapid water stress via abscisic acid (ABA)-dependent or -independent pathways [late embryogenesis abundant (LEA)-like and heat shock cognate (HS) 70, respectively], or in response to pathogens or oxidative stress [β-1,3 glucanase (BG), annexin] and (ii) metabolic enzymes [glutamine synthetase (GS), fructokinase (Frk), caffeoyl-CoA-3-O-methyltransferase (CCoAOMT)]. The differential behaviour of these proteins highlighted a role for AXR2 and/or AXR1 in the regulation of their abundance during drought adaptation. In particular, reduced induction of RD29B, GS and annexin, and overexpression of BG2 were observed specifically in the axr1-3 mutant, which is dramatically affected in several ABA-dependent drought adaptive responses, such as drought rhizogenesis. Altogether these results indicate cross-talk between auxin- and ABA-signalling in Arabidopsis drought responses