RESUMO
Multiple endocrine neoplasia syndrome type 1 (MEN1), which is secondary to mutation of the MEN1 gene, is a rare autosomal-dominant disease that predisposes mutation carriers to endocrine tumors. Although genotype-phenotype studies have so far failed to identify any statistical correlations, some families harbor recurrent tumor patterns. The function of MENIN is unclear, but has been described through the discovery of its interacting partners. Mutations in the interacting domains of MENIN functional partners have been shown to directly alter its regulation abilities. We report on a cohort of MEN1 patients from the Groupe d'étude des Tumeurs Endocrines. Patients with a molecular diagnosis and a clinical follow-up, totaling 262 families and 806 patients, were included. Associations between mutation type, location or interacting factors of the MENIN protein and death as well as the occurrence of MEN1-related tumors were tested using a frailty Cox model to adjust for potential heterogeneity across families. Accounting for the heterogeneity across families, the overall risk of death was significantly higher when mutations affected the JunD interacting domain (adjusted HR = 1.88: 95%-CI = 1.15-3.07). Patients had a higher risk of death from cancers of the MEN1 spectrum (HR = 2.34; 95%-CI = 1.23-4.43). This genotype-phenotype correlation study confirmed the lack of direct genotype-phenotype correlations. However, patients with mutations affecting the JunD interacting domain had a higher risk of death secondary to a MEN1 tumor and should thus be considered for surgical indications, genetic counseling and follow-up.
Assuntos
Neoplasia Endócrina Múltipla Tipo 1/genética , Neoplasia Endócrina Múltipla Tipo 1/mortalidade , Mutação , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas/genética , Família , Feminino , Seguimentos , Humanos , Masculino , Neoplasia Endócrina Múltipla Tipo 1/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fatores de RiscoRESUMO
In the allopolyploid Brassica napus, we obtained a petal-closed flower mutation by ethyl methanesulfonate mutagenesis. Here, we report cloning and characterization of the Bn-CLG1A (CLG for cleistogamy) gene and the Bn-clg1A-1D mutant allele responsible for the cleistogamy phenotype. Bn-CLG1A encodes a RINGv E3 ubiquitin ligase that is highly conserved across eukaryotes. In the Bn-clg1A-1D mutant allele, a C-to-T transition converts a Pro at position 325 to a Leu (P325L), causing a dominant mutation leading to cleistogamy. B. napus and Arabidopsis thaliana plants transformed with a Bn-clg1A-1D allele show cleistogamous flowers, and characterization of these flowers suggests that the Bn-clg1A-1D mutation causes a pronounced negative regulation of cutin biosynthesis or loading and affects elongation or differentiation of petal and sepal cells. This results in an inhibition or a delay of petal development, leading to folded petals. A homoeologous gene (Bn-CLG1C), which shows 99.5% amino acid identity and is also constitutively and equally expressed to the wild-type Bn-CLG1A gene, was also identified. We showed that P325L is not a loss-of-function mutation and did not affect expression of Bn-clg1A-1D or Bn-CLG1C. Our findings suggest that P325L is a gain-of-function semidominant mutation, which led to either hyper- or neofunctionalization of a redundant homoeologous gene.
Assuntos
Brassica napus/metabolismo , Proteínas de Plantas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Brassica napus/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Dados de Sequência Molecular , Proteínas de Plantas/genética , Mutação Puntual/genética , Mutação Puntual/fisiologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
It has frequently been hypothesized that quantitative resistance increases the durability of qualitative (R-gene mediated) resistance but supporting experimental evidence is rare. To test this hypothesis, near-isogenic lines with/without the R-gene Rlm6 introduced into two Brassica napus cultivars differing in quantitative resistance to Leptosphaeria maculans were used in a 5-yr field experiment. Recurrent selection of natural fungal populations was done annually on each of the four plant genotypes, using crop residues from each genotype to inoculate separately the four series of field trials for five consecutive cropping seasons. Severity of phoma stem canker was measured on each genotype and frequencies of avirulence alleles in L. maculans populations were estimated. Recurrent selection of virulent isolates by Rlm6 in a susceptible background rendered the resistance ineffective by the third cropping season. By contrast, the resistance was still effective after 5 yr of selection by the genotype combining this gene with quantitative resistance. No significant variation in the performance of quantitative resistance alone was noted over the course of the experiment. We conclude that quantitative resistance can increase the durability of Rlm6. We recommend combining quantitative resistance with R-gene mediated resistance to enhance disease control and crop production.
Assuntos
Ascomicetos/genética , Ascomicetos/patogenicidade , Brassica napus/genética , Brassica napus/microbiologia , Genes de Plantas , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Alelos , Proteínas de Transporte/genética , Produtos Agrícolas/genética , Produtos Agrícolas/microbiologia , GenótipoRESUMO
As part of a research programme focused on flavonoid biosynthesis in the seed coat of Brassica napus L. (oilseed rape), orthologs of the BANYULS gene that encoded anthocyanidin reductase were cloned in B. napus as well as in the related species Brassica rapa and Brassica oleracea. B. napus genome contained four functional copies of BAN, two originating from each diploid progenitor. Amino acid sequences were highly conserved between the Brassicaceae including B. napus, B. rapa, B. oleracea as well as the model plant Arabidopsis thaliana. Along the 200 bp in 5' of the ATG codon, Bna.BAN promoters (ProBna.BAN) were conserved with AtANR promoter and contained putative cis-acting elements. In addition, transgenic Arabidopsis and oilseed rape plants carrying the first 230 bp of ProBna.BAN fused to the UidA reporter gene were generated. In the two Brassicaceae backgrounds, ProBna.BAN activity was restricted to the seed coat. In B. napus seed, ProBna.BAN was activated in procyanidin-accumulating cells, namely the innermost layer of the inner integument and the micropyle-chalaza area. At the transcriptional level, the four Bna.BAN genes were expressed in the seed. Laser microdissection assays of the seed integuments showed that Bna.BAN expression was restricted to the inner integument, which was consistent with the activation profile of ProBna.BAN. Finally, Bna.BAN genes were mapped onto oilseed rape genetic maps and potential co-localisations with seed colour quantitative trait loci are discussed.
Assuntos
Biflavonoides/metabolismo , Brassica/genética , Catequina/metabolismo , NADH NADPH Oxirredutases/genética , Proteínas de Plantas/genética , Proantocianidinas/metabolismo , Sementes/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Brassica/enzimologia , Brassica/metabolismo , Brassica napus/enzimologia , Brassica napus/genética , Brassica napus/metabolismo , Brassica rapa/enzimologia , Brassica rapa/genética , Brassica rapa/metabolismo , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Perfilação da Expressão Gênica , Genoma de Planta , Glucuronidase/genética , Glucuronidase/metabolismo , Dados de Sequência Molecular , Família Multigênica , NADH NADPH Oxirredutases/classificação , NADH NADPH Oxirredutases/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sementes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
As part of an ongoing research program dedicated to the understanding of proanthocyanidin (PA) accumulation in Brassica napus seed coat, transgenic rapeseed plants carrying a 2.3-kb fragment of the Arabidopsis thaliana BAN promoter (ProAtBAN) fused to the uidA reporter gene (GUS) were generated. Analysis of these plants revealed that ProAtBAN was activated in B. napus seed coat, following a spatio-temporal pattern that was very similar to the PA deposition profile in rapeseed and also to the one previously described in Arabidopsis. ProAtBAN activity occurred as soon as the early stages of embryogenesis and was restricted to the cells where PAs were shown to accumulate. Therefore, the Arabidopsis BAN promoter can be used to trigger gene expression in B. napus seed coat for both genetic engineering and functional validation of candidate genes. In addition, these data strongly suggest that the transcriptional regulatory network of the BAN gene is conserved between Arabidopsis and rapeseed. This is consistent with the fact that similarity searches of the public rapeseed sequence databases allowed recovering the rapeseed homologs for several BAN regulators, namely TT1, TT2, TT8, TT16 and TTG1, which have been previously described in Arabidopsis.
Assuntos
Brassica napus/metabolismo , Proantocianidinas/metabolismo , Regiões Promotoras Genéticas , Sementes/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassica napus/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Oilseed rape (Brassica napus L.) is a major oil crop that also supplies proteins for the feed industry. In order to reduce total cost production, the objective is to increase oil yield while reducing crop inputs (especially nitrogen and pesticides). Concomitantly, it is necessary to anticipate specific uses (e.g., fatty acid composition) and to ensure the valorisation of the by-products (rapeseed meal). By the past, improvement of seed quality focused on fatty acid balance and low seed glucosinolate content. Current goals include the breeding of yellow-seeded rapeseed lines with high content of seed oil. The use of molecular tools and the exploitation of Arabidopsis knowledge will be presented and discussed.
Assuntos
Brassica napus/genética , Cruzamento/métodos , Sementes/química , Ração Animal/análise , Animais , Brassica napus/metabolismo , Ácidos Graxos/metabolismo , Alimentos Geneticamente Modificados , Genes de Plantas , Variação Genética , Glucosinolatos/efeitos adversos , Glucosinolatos/metabolismo , Valor Nutritivo , Óleos de Plantas/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Ratos , Sementes/metabolismo , Triglicerídeos/metabolismoRESUMO
Modern approaches to minimally invasive ablative treatment of solid tumors involve the use of miniature instruments and combined treatments. These can be enhanced with ultrasound imaging that depicts tumor margins; facilitates guidance, delivery, and dosage of local chemotherapy; and can monitor the effectiveness of the treatment. This paper describes the advantages of ultrasound guided cryosurgery combined with local chemotherapy delivered in multilamellar, echogenic microcapsules of 5-FU ("microcaps") using a xenograft tumor model. Genetically engineered bioluminescent human prostate tumor cells, DU-145(Luc+), were implanted subcutaneously into athymic nude mice. Experiments were designed to mimic the situation where palliative cryoablation spares a portion of the tumor so that the combined effect of cryosurgery and focal injections of chemotherapeutic microcapsules could be evaluated. Eighteen (18) tumors were treated with percutaneous partial cryoablation or interstitial chemoablation, or a combination of both. A single F/T cycle was applied to tumor and micro-encapsulated chemotherapy is delivered at outer margin of frozen tumor in two opposite sites. Results show that the tumor and cryosurgical kill zone contours were seen with both the bio-luminescence assay (BLI) and ultrasonography (US). US can easily detect as little as 2 microl of echogenic microcaps, and monitor their lifetime in the tumor tissue. BLI was determinant in showing that minute amounts of microcapsule chemotherapy (38.7 ng of 5-FU/g tumor) dramatically inhibited tumor growth starting within two days after injection. The mean BLI emitted by control tumors was 5.6 times greater at Day 4 than the BLI measurements from tumors treated with 5-FU microcaps (p=0.036). By Day 7, BLI values from the control tumors were still 2.7 times greater than those treated with 5-FU microcaps (p<0.01). In tumors treated by partial cryoablation, the mean BLI of viable tumor cells was 20 times less at day 3 (p=0.05) and 46% less at day 7 than the non-treated tumors. The combined treatment produced a dramatic inhibition of tumor growth that lasted throughout the 7-day study. The BLI measured from viable tumor cells in non-treated tumors was 34 times greater at day 3 and more than 350 times greater at day 7 than those treated by combined cryoablation and 5-FU microcaps. The results demonstrated, for the first time, that a single moderate freeze of a human prostate tumor combined with bi-focal peripheral microcapsule chemotherapy (5-FU) has a better and longer inhibitory effect on tumor growth compared to the growth inhibition rendered by cryosurgery or local microcapsule chemo-therapy alone. This shows promise for a new, focal, combined ablative modality using US guided deposition of microencapsulated drug(s) and echogenic markers deposited in the hypothermic margin of tumors which could enhance the efficacy of cryoablation of prostate cancers.
Assuntos
Criocirurgia , Fluoruracila/uso terapêutico , Medições Luminescentes , Neoplasias da Próstata/prevenção & controle , Animais , Terapia Combinada , Diagnóstico por Imagem , Engenharia Genética , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Biológicos , Monitorização Intraoperatória , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/tratamento farmacológico , Transplante Heterólogo , Resultado do Tratamento , Células Tumorais Cultivadas , UltrassonografiaRESUMO
Proanthocyanidins (PAs) are seed coat flavonoids that impair the digestibility of Brassica napus meal. Development of low-PA lines is associated with a high-quality meal and with increased contents in oil and proteins, but requires better knowledge of seed flavonoids. Flavonoids in Brassica mature seed are mostly insoluble so that very few qualitative and quantitative data are available yet. In the present study, the profiling of seed coat flavonoids was established in eight black-seeded B. napus genotypes, during seed development when soluble flavonoids were present and predominated over the insoluble forms. Thirteen different flavonoids including (-)-epicatechin, five procyanidins (PCs which are PAs composed of epicatechin oligomers only) and seven flavonols (quercetin-3-O-glucoside, quercetin-dihexoside, isorhamnetin-3-O-glucoside, isorhamnetin-hexoside-sulfate, isorhamnetin-dihexoside, isorhamnetin-sinapoyl-trihexoside and kaempferol-sinapoyl-trihexoside) were identified and quantified using liquid chromatography coupled to electrospray ionization-mass spectrometry (LC-ESI-MS(n)). These flavonol derivatives were characterized for the first time in the seed coat of B. napus, and isorhamnetin-hexoside-sulfate and isorhamnetin-sinapoyl-trihexoside were newly identified in Brassica spp. High amounts of PCs accumulated in the seed coat, with solvent-soluble polymers of (-)-epicatechin reaching up to 10% of the seed coat weight during seed maturation. In addition, variability for both PC and flavonol contents was observed within the panel of eight black-seeded genotypes. Our results provide new insights into breeding for low-PC B. napus genotypes.
Assuntos
Brassica napus/química , Flavonoides/análise , Sementes/química , Sementes/crescimento & desenvolvimento , Brassica napus/genética , Catequina/análise , Cromatografia Líquida , Flavonóis/análise , Genótipo , Cinética , Proantocianidinas/análise , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Near-isogenic Brassica napus lines carrying/lacking resistance gene Rlm6 were used to investigate the effects of temperature and leaf wetness duration on phenotypic expression of Rlm6-mediated resistance. Leaves were inoculated with ascospores or conidia of Leptosphaeria maculans carrying the effector gene AvrLm6. Incubation period to the onset of lesion development, number of lesions and lesion diameter were assessed. Symptomless growth of L. maculans from leaf lesions to stems was investigated using a green fluorescent protein (GFP) expressing isolate carrying AvrLm6. L. maculans produced large grey lesions on Darmor (lacking Rlm6) at 5-25 degrees C and DarmorMX (carrying Rlm6) at 25 degrees C, but small dark spots and 'green islands' on DarmorMX at 5-20 degrees C. With increasing temperature/wetness duration, numbers of lesions/spots generally increased. GFP-expressing L. maculans grew from leaf lesions down leaf petioles to stems on DarmorMX at 25 degrees C but not at 15 degrees C. We conclude that temperature and leaf wetness duration affect the phenotypic expression of Rlm6-mediated resistance in leaves and subsequent L. maculans spread down petioles to produce stem cankers.
Assuntos
Ascomicetos , Brassica napus/genética , Brassica napus/microbiologia , Doenças das Plantas/microbiologia , Temperatura , Ascomicetos/crescimento & desenvolvimento , Brassica napus/anatomia & histologia , Cruzamentos Genéticos , Genes de Plantas , Imunidade Inata/genética , Modelos Biológicos , Fenótipo , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Caules de Planta/anatomia & histologia , Caules de Planta/genética , Caules de Planta/microbiologia , Fatores de Tempo , ÁguaRESUMO
The radish Rfo gene restores male fertility in radish or rapeseed plants carrying Ogura cytoplasmic male-sterility. This system was first discovered in radish and was transferred to rapeseed for the production of F1 hybrid seeds. We aimed to identify the region of the Arabidopsis genome syntenic to the Rfo locus and to characterize the radish introgression in restored rapeseed. We used two methods: amplified consensus genetic markers (ACGMs) in restored rapeseed plants and construction of a precise genetic map around the Rfo gene in a segregating radish population. The use of ACGMs made it possible to detect radish orthologs of Arabidopsis genes in the restored rapeseed genome. We identified radish genes, linked to Rfo in rapeseed and whose orthologs in Arabidopsis are carried by chromosomes 1, 4 and 5. This indicates several breaks in colinearity between radish and Arabidopsis genomes in this region. We determined the positions of markers relative to each other and to the Rfo gene, using the progeny of a rapeseed plant with unstable meiotic transmission of the radish introgression. This enabled us to produce a schematic diagram of the radish introgression in rapeseed. Markers which could be mapped both on radish and restored rapeseed indicate that at least 50 cM of the radish genome is integrated in restored rapeseed. Using markers closely linked to the Rfo gene in rapeseed and radish, we identified a contig spanning six bacterial artificial chromosome (BAC) clones on Arabidopsis chromosome 1, which is likely to carry the orthologous Rfo gene.
Assuntos
Arabidopsis/genética , Brassica/genética , Genoma de Planta , Proteínas de Plantas/genética , Arabidopsis/fisiologia , Cromossomos Artificiais Bacterianos , Reação em Cadeia da PolimeraseRESUMO
Ogura cytoplasmic male sterility (CMS) in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene, Orf138, that prevents the production of functional pollen without affecting female fertility. Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the post-transcriptional level. The Ogura CMS/Rfo two-component system is a useful model for investigating nuclear-cytoplasmic interactions, as well as the physiological basis of fertility restoration. Using a combination of positional cloning and microsynteny analysis of Arabidopsis thaliana and radish, we genetically and physically delimited the Rfo locus to a 15-kb DNA segment. Analysis of this segment shows that Rfo is a member of the pentatricopeptide repeat (PPR) family. In Arabidopsis, this family contains more than 450 members of unknown function, although most of them are predicted to be targeted to mitochondria and chloroplasts and are thought to have roles in organellar gene expression.