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1.
Genet Mol Res ; 15(3)2016 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-27525929

RESUMO

Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance.


Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Digitaria/enzimologia , Glicina/análogos & derivados , Herbicidas/farmacologia , Proteínas de Plantas/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/metabolismo , Substituição de Aminoácidos , Digitaria/efeitos dos fármacos , Digitaria/genética , Expressão Gênica , Glicina/farmacologia , Resistência a Herbicidas , Filogenia , Proteínas de Plantas/metabolismo , Polimorfismo Genético , Análise de Sequência de DNA
2.
J Environ Monit ; 14(8): 2108-13, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22760469

RESUMO

Persistent harmful scenarios associated with disposal of radioactive waste, high-background radiation areas and severe nuclear accidents are of great concern regarding consequences to both human health and the environment. Of particular concern is the extracellular DNA in aquatic environments contaminated by radiological substances. Strand breaks induced by radiation promote decrease in the transformation efficiency for extracellular DNA. The focus of this study is the quantification of DNA damage following long-term exposure (over one year) to low doses of natural uranium (an alpha particle emitter) to simulate natural conditions, since nothing is known about alpha radiation induced damage to extracellular DNA. A high-resolution Atomic Force Microscope was used to evaluate DNA fragments. Double-stranded plasmid pBS as a model for extracellular DNA was exposed to different amounts of natural uranium. It was demonstrated that low concentrations of U in water (50 to 150 ppm) produce appreciable numbers of double strand breaks, scaling with the square of the average doses. The importance of these findings for environment monitoring of radiological pollution is addressed.


Assuntos
Fragmentação do DNA/efeitos da radiação , DNA/efeitos da radiação , Urânio/toxicidade , Poluentes Radioativos da Água/toxicidade , Dano ao DNA , Urânio/análise , Poluentes Radioativos da Água/análise
3.
Genet Mol Res ; 10(3): 2024-33, 2011 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-21948764

RESUMO

Microsatellites, or simple sequence repeats (SSRs), and their flanking regions in chloroplast genomes (plastomes) of some species of the family Poaceae were analyzed in silico to look for DNA sequence variations. Comparison of the complete chloroplast DNA sequences (cpDNAs) of sugarcane (Saccharum hybrid cv. SP-80-3280 and S. officinarum cv. NCo310) and related species, Agrostis stolonifera, Brachypodium distachyon, Hordeum vulgare subsp vulgare, Lolium perenne, Oryza nivara, O. sativa subsp indica, O. sativa subsp japonica, Sorghum bicolor, Triticum aestivum, Zea mays, and Z. mays cv. B73, allowed us to examine the organization of chloroplast SSRs (cpSSRs) in genic and intergenic regions. We identified 204 cpSSRs in the sugarcane cpDNA; 22.5% were in genic regions. The ndh, rps, trn, and rpl gene clusters of the chloroplasts had the most repeats. Mononucleotide repeats were the most abundant cpSSRs in these species; however, di-, tri-, tetra-, penta-, and hexanucleotide repeats were also identified. Many base substitutions and deletions/insertions were identified in the cpSSR loci and their flanking regions. Multiple alignments of all cpSSR sequences of Poaceae species made identification of nucleotide variability possible; repeat motifs are not uniformly distributed across the Poaceae plastomes, but are mostly confined to intergenic regions. Phylogeny was determined by maximum parsimony and neighbor-joining inference methods. The cpSSRs of these species were found to be polymorphic. It appears that individual cpSSRs in the Poaceae are stable, at least over short periods of evolutionary time. We conclude that the plastome database can be exploited for phylogenetic analysis and biotechnological development.


Assuntos
DNA de Cloroplastos/genética , Repetições de Microssatélites/genética , Saccharum/genética , Sequência de Bases , Cloroplastos/genética , Marcadores Genéticos , Variação Genética , Genoma de Cloroplastos , Genoma de Planta , Filogenia , Polimorfismo Genético , Alinhamento de Sequência , Análise de Sequência de DNA
4.
Genet Mol Res ; 9(3): 1298-311, 2010 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-20623455

RESUMO

The pathogenic fungus Fusarium graminearum is an ongoing threat to agriculture, causing losses in grain yield and quality in diverse crops. Substantial progress has been made in the identification of genes involved in the suppression of phytopathogens by antagonistic microorganisms; however, limited information regarding responses of plant pathogens to these biocontrol agents is available. Gene expression analysis was used to identify differentially expressed transcripts of the fungal plant pathogen F. graminearum under antagonistic effect of the bacterium Pantoea agglomerans. A macroarray was constructed, using 1014 transcripts from an F. graminearum cDNA library. Probes consisted of the cDNA of F. graminearum grown in the presence and in the absence of P. agglomerans. Twenty-nine genes were either up (19) or down (10) regulated during interaction with the antagonist bacterium. Genes encoding proteins associated with fungal defense and/or virulence or with nutritional and oxidative stress responses were induced. The repressed genes coded for a zinc finger protein associated with cell division, proteins containing cellular signaling domains, respiratory chain proteins, and chaperone-type proteins. These data give molecular and biochemical evidence of response of F. graminearum to an antagonist and could help develop effective biocontrol procedures for pathogenic plant fungi.


Assuntos
Antibiose/genética , Produtos Agrícolas/microbiologia , Fusarium/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Pantoea/fisiologia , Regulação para Baixo/genética , Fusarium/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/genética
5.
J Neuroendocrinol ; 21(7): 629-39, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19490365

RESUMO

The noradrenergic nucleus locus coeruleus (LC) has been reported to regulate luteinising hormone (LH) secretion in female rats. Both oestrogen and progestin receptors have been demonstrated in LC neurones, suggesting that these cells are possibly responsive to variations in circulating levels of ovarian steroids. We therefore evaluated changes in the activity of LC neurones during the oestrous cycle and after ovarian-steroid treatment in ovariectomised (OVX) rats, as determined by immunoreactivity to Fos-related antigens (FRA), which comprises all of the known members of the Fos family. Effects of ovarian steroids on the firing rate of LC neurones were also determined in a slice preparation. The number of FRA/tyrosine hydroxylase (TH)-immunoreactive (ir) neurones in the LC increased from 14.00-16.00 h on pro-oestrus, coinciding with the onset of the LH surge and rise in plasma progesterone. FRA immunoreactivity was unaltered during dioestrus. Oestradiol-treated OVX rats (OVX+E) displayed marked reduction in FRA/TH-ir neurones in LC compared to oil-treated OVX rats. Accordingly, oestradiol superfusion significantly reduced the spontaneous firing rate of LC neurones in slices from OVX rats. Compared to OVX+E, oestradiol-treated rats injected with progesterone at 08.00 h (OVX+EP) exhibited higher number of FRA/TH-ir neurones in the LC at 10.00 h and 16.00 h, and great amplification of the LH surge. Bath application of progesterone significantly increased the spontaneous firing rate of OVX+E LC neurones. Our data suggest that ovarian steroids may physiologically modulate the activity of LC neurones in females, with possible implications for LH secretion. Moreover, oestradiol and progesterone appear to exert opposite and complementary effects (i.e. whereas oestradiol inhibits, progesterone, after oestradiol priming, stimulates LC activity).


Assuntos
Estrogênios/metabolismo , Ciclo Estral/fisiologia , Locus Cerúleo/fisiologia , Hormônio Luteinizante/metabolismo , Neurônios/fisiologia , Progesterona/metabolismo , Potenciais de Ação , Animais , Estradiol/farmacologia , Estrogênios/farmacologia , Feminino , Técnicas In Vitro , Locus Cerúleo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ovariectomia , Progesterona/sangue , Progesterona/farmacologia , Progestinas/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismo
6.
J Steroid Biochem Mol Biol ; 93(2-5): 319-23, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15860276

RESUMO

Previous work from our laboratory has shown that in cultures of hypothalamic neurons obtained from male fetuses at embryonic day 16 the axogenic response to estradiol (E2) is contingent upon culture with medium conditioned by astroglia from a target region for hypothalamic axons. E2 also induced increased levels of TrkB that were necessary for the axonal growth to occur. This convergence between estrogenic and neurotrophic signals prompted investigation of the mitogen activated protein kinase (MAPK) cascade. Analysis of the temporal course of MAPK activation showed increased levels of phosphorylated ERK up to 60 min after E2 exposure, with a maximal response at 5-15 min. UO126 (specific inhibitor of MEK 1/2) blocked E2 induced axonal elongation and ERK phosphorylation, confirming the involvement of ERK in the neuritogenic effect of E2. The membrane impermeable construct E2-BSA proved as effective as free E2 to induce axon elongation, suggesting that E2 exerted its effect through a membrane-associated receptor. This possibility received additional support from experiments showing that E2-BSA also increased ERK phosphorylation with the same time course than E2. These results indicate that ERK signaling is necessary for E2 to induce axon growth and this activation is mediated by a membrane bound estrogen receptor.


Assuntos
Estradiol/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Feminino , Feto/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Neurônios/metabolismo , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Gravidez , Ratos , Receptor trkB/antagonistas & inibidores , Receptor trkB/genética , Diferenciação Sexual/efeitos dos fármacos
7.
Eur J Neurosci ; 20(2): 331-7, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15233742

RESUMO

17-beta-estradiol (E2) increases axonal growth and tyrosine kinase receptor (Trk)B levels of male-derived hypothalamic neurones in vitro. To investigate whether the axogenic response depends on the upregulation of TrkB, we analysed neuritic growth and neuronal polarization in cultures treated with an antisense oligonucleotide against TrkB mRNA. In cultures without E2, treatment with 7.5 or 10 micro m antisense reduced TrkB levels and the percentage of neurones showing an identifiable axon; the number and length of minor processes were increased. In cultures treated with 5 micro m antisense, morphometric parameters were normal although total TrkB levels were reduced. The same dose prevented the E2-dependent increase of TrkB levels and suppressed the axogenic effect of E2. These results indicate that TrkB is necessary for normal neuronal growth and maturation and further suggest that an increase in TrkB is necessary for E2 to exert its axogenic effect in male-derived neurones.


Assuntos
Estradiol/farmacologia , Hipotálamo/citologia , Neurônios/efeitos dos fármacos , Receptor trkB/metabolismo , Animais , Western Blotting/métodos , Contagem de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Imuno-Histoquímica/métodos , Técnicas In Vitro , Masculino , Neuritos/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neurônios/fisiologia , Oligonucleotídeos Antissenso/farmacologia , Ratos , Ratos Wistar , Receptor trkB/antagonistas & inibidores , Receptor trkB/genética , Fatores de Tempo
8.
J Bacteriol ; 186(7): 2164-72, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15028702

RESUMO

Leptospira species colonize a significant proportion of rodent populations worldwide and produce life-threatening infections in accidental hosts, including humans. Complete genome sequencing of Leptospira interrogans serovar Copenhageni and comparative analysis with the available Leptospira interrogans serovar Lai genome reveal that despite overall genetic similarity there are significant structural differences, including a large chromosomal inversion and extensive variation in the number and distribution of insertion sequence elements. Genome sequence analysis elucidates many of the novel aspects of leptospiral physiology relating to energy metabolism, oxygen tolerance, two-component signal transduction systems, and mechanisms of pathogenesis. A broad array of transcriptional regulation proteins and two new families of afimbrial adhesins which contribute to host tissue colonization in the early steps of infection were identified. Differences in genes involved in the biosynthesis of lipopolysaccharide O side chains between the Copenhageni and Lai serovars were identified, offering an important starting point for the elucidation of the organism's complex polysaccharide surface antigens. Differences in adhesins and in lipopolysaccharide might be associated with the adaptation of serovars Copenhageni and Lai to different animal hosts. Hundreds of genes encoding surface-exposed lipoproteins and transmembrane outer membrane proteins were identified as candidates for development of vaccines for the prevention of leptospirosis.


Assuntos
Genoma Bacteriano , Genômica , Leptospira interrogans/fisiologia , Leptospira interrogans/patogenicidade , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cricetinae , Humanos , Leptospira interrogans/classificação , Leptospira interrogans/genética , Leptospirose/microbiologia , Camundongos , Dados de Sequência Molecular , Análise de Sequência de DNA , Sorotipagem , Virulência/genética
10.
J Bacteriol ; 185(3): 1018-26, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12533478

RESUMO

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.


Assuntos
Citrus/microbiologia , Gammaproteobacteria/genética , Genoma Bacteriano , Doenças das Plantas/microbiologia , Sequência de Bases , Dados de Sequência Molecular
11.
Ann N Y Acad Sci ; 1007: 306-16, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14993063

RESUMO

Previous work from our laboratory has shown that in cultures of hypothalamic neurons obtained from male fetuses at embryonic day 16, the axogenic response to estrogen (E2) is contingent on coculture with target glia or target glia-conditioned media (CM). Neither the estrogen receptor blockers tamoxifen nor ICI 182,780 prevented the axogenic effects of the hormone. Estradiol made membrane-impermeable by conjugation to a protein of high molecular weight (E2-BSA) preserved its axogenic capacity, suggesting the possibility of a membrane effect responsible for the action of E2. Western blot analysis of extracts from homogenates of cultured neurons grown with E2 and CM from target glia had more TrkB than cultures with CM alone or E2 alone. To further investigate the interaction between E2 and the neurotrophin receptors, we used a specific antisense oligonucleotide (AS) to prevent the estradiol-induced increase of TrkB. The effect of E2 was suppressed in cultures in which TrkB was down-regulated by the AS, showing decreased axonal elongation when compared with neurons treated with E2 without AS or with sense TrkB. In cultures grown with AS, the axonal length of E2-treated cultures was not different from cultures without E2. Evidence suggesting cross-talk between E2 and neurotrophic factor(s) prompted investigation of signaling along the MAPK cascade. Immuno blotting of E2-treated cultures showed increased levels of phosphorylated ERK1 and ERK2. UO126 but not LY294002 blocked E2-induced axonal elongation, suggesting that the MAPKs are involved in this response.


Assuntos
Axônios/metabolismo , Estradiol/metabolismo , Hipotálamo/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Axônios/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Antagonistas de Hormônios/farmacologia , Humanos , Hipotálamo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo
12.
Braz J Med Biol Res ; 35(6): 645-50, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12045828

RESUMO

The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.


Assuntos
Aderência Bacteriana/fisiologia , Cálcio/metabolismo , Citrus/microbiologia , Gammaproteobacteria/fisiologia , Doenças das Plantas/microbiologia , Compostos de Sulfidrila/metabolismo , Cálcio/química , Citrus/química , Microanálise por Sonda Eletrônica , Gammaproteobacteria/genética , Gammaproteobacteria/ultraestrutura , Genoma Bacteriano , Magnésio/química , Metionina Sulfóxido Redutases , Oxirredutases/química , Oxirredutases/metabolismo , Compostos de Sulfidrila/química , Enxofre/química
13.
Braz. j. med. biol. res ; 35(6): 645-650, June 2002. ilus
Artigo em Inglês | LILACS | ID: lil-309520

RESUMO

The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls


Assuntos
Cálcio , Citrus , Gammaproteobacteria , Doenças das Plantas , Compostos de Sulfidrila , Cálcio , Adesão Celular , Agregação Celular , Citrus , Microanálise por Sonda Eletrônica , Gammaproteobacteria , Genoma Bacteriano , Magnésio , Oxirredutases , Compostos de Sulfidrila , Enxofre
14.
J Neurosci Res ; 66(3): 475-81, 2001 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-11746365

RESUMO

The purpose of the present work was to investigate the participation of estradiol receptors (ER) in estrogen-induced axon growth in vitro. Hypothalamic neurons from 16 day (E16) male rat fetuses were cultured with or without 17-beta-estradiol at 1 x 10(-7) M in basal medium or medium conditioned by astroglia derived from ventral mesencephalon (CM). After 48 hr in vitro, neurite outgrowth was quantified by morphometric analysis. An axogenic effect could be demonstrated for estradiol added to CM. With RT-PCR, the mRNA transcript for ERalpha was found in the donor tissues as well as in the neuron cultures. In this model two specific nuclear ER blockers (tamoxifen and ICI 182,780) were ineffective in blocking the neuritogenic effect, and a membrane-impermeable estrogen-albumin construct (E2-BSA) was as effective as estradiol. These results indicate that the axogenic effect of estradiol at E16 is not exerted through the classical intracellular receptor signal transduction system and suggest the possibility of a membrane-mediated mechanism. The data are discussed in light of our previous findings pointing to the interdependent activation of the estrogenic and the trophic factor signaling pathways that mediate stimulated axon growth.


Assuntos
Diferenciação Celular/fisiologia , Estradiol/metabolismo , Cones de Crescimento/metabolismo , Hipotálamo/embriologia , Fatores de Crescimento Neural/metabolismo , Receptores de Estrogênio/genética , Transdução de Sinais/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Feto , Genoma , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/ultraestrutura , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Masculino , Fatores de Crescimento Neural/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Receptores de Estrogênio/antagonistas & inibidores , Reprodução/efeitos dos fármacos , Reprodução/fisiologia , Soroalbumina Bovina/farmacologia , Fatores Sexuais , Transdução de Sinais/efeitos dos fármacos
15.
Proc Natl Acad Sci U S A ; 98(21): 12103-8, 2001 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-11593022

RESUMO

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.


Assuntos
Etiquetas de Sequências Expressas , Genoma Humano , Fases de Leitura Aberta , Transcrição Genética , Humanos
16.
Nat Biotechnol ; 19(9): 870-5, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11533648

RESUMO

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.


Assuntos
Biotecnologia/métodos , Plantas Geneticamente Modificadas , Plastídeos/genética , Transformação Genética , Transgenes , Cloroplastos/metabolismo , Immunoblotting , Modelos Genéticos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Fatores de Tempo
17.
Proc Natl Acad Sci U S A ; 97(23): 12690-3, 2000 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-11070084

RESUMO

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).


Assuntos
Cromossomos Humanos Par 22 , Transcrição Genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Humanos , Fases de Leitura Aberta
18.
Eur J Neurosci ; 12(7): 2291-8, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10947808

RESUMO

To determine whether soluble products from different CNS regions differ in their ability to support oestrogen-stimulated neurite growth, hypothalamic neurons from sexually segregated embryos were cultured with astroglia-conditioned medium (CM) derived from cortex, striatum and mesencephalon, with or without 17-beta-oestradiol 100 nM added to the medium. After 48 h in vitro, neurite outgrowth was quantified by morphometric analysis. Astroglia-CM from mesencephalon (a target for the axons of hypothalamic neurons) induced the greatest axogenic response in males and in this case only a neuritogenic effect could be demonstrated for oestradiol. On the other hand, astroglia-CM from regions that do not receive projections from ventromedial hypothalamus inhibited axon growth. A sexual difference in the response of hypothalamic neurons to astroglia-CM and oestradiol was found; growth of neurons from female foetuses was increased by astroglia-CM from mesencephalon, but no neuritogenic effect could be demonstrated for oestradiol in these cultures. Blot immunobinding demonstrated the presence of receptors for neurotrophic factors in cultures of hypothalamic neurons; Western blot analysis of these cultures demonstrated that oestradiol increased the concentration of trkB and IGF-I Rbeta, whereas trkA was not detected and the concentration of trkC was not modified. These results support the hypothesis that target regions produce some factor(s) that stimulate the growth of axons from projecting neurons and further indicate that in the case of males this effect is modulated by oestradiol, perhaps mediated through the upregulation of trkB and IGF-I receptors.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Estradiol/farmacologia , Neurônios/efeitos dos fármacos , Caracteres Sexuais , Núcleo Hipotalâmico Ventromedial/citologia , Animais , Astrócitos/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Immunoblotting , Masculino , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurônios/química , Neurônios/ultraestrutura , Gravidez , Ratos , Ratos Wistar , Receptor trkA/análise , Receptor trkB/análise , Receptor trkC/análise
19.
Nature ; 406(6792): 151-9, 2000 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-10910347

RESUMO

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.


Assuntos
Genoma Bacteriano , Plantas/microbiologia , Pseudomonadaceae/genética , Análise de Sequência de DNA , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Transporte Biológico , Mapeamento Cromossômico , Citrus/microbiologia , Reparo do DNA , DNA Bacteriano , Metabolismo Energético , Dados de Sequência Molecular , Plantas Tóxicas , Biossíntese de Proteínas , Pseudomonadaceae/metabolismo , Pseudomonadaceae/patogenicidade , Tabaco/microbiologia , Transcrição Genética , Virulência/genética
20.
Plant J ; 22(2): 97-104, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10792825

RESUMO

The chloroplast genomes of most higher plants contain two giant open reading frames designated ycf1 and ycf2. In tobacco, ycf1 potentially specifies a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the functions of ycf1 and ycf2, we have constructed several mutant alleles for targeted disruption and/or deletion of these two reading frames. The mutant alleles were introduced into the tobacco plastid genome by biolistic chloroplast transformation to replace the corresponding wild-type alleles by homologous recombination. Chloroplast transformants were obtained for all constructs and tested for their homoplastomic state. We report here that all transformed lines remained heteroplastomic even after repeated cycles of regeneration under high selective pressure. A balanced selection was observed in the presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild-type versus transformed genome copies. Upon removal of the antibiotic and therewith release of the selective pressure, sorting out towards the wild-type plastid genome occurred in all transplastomic lines. These findings suggest that ycf1 and ycf2 are functional genes and encode products that are essential for cell survival. The two reading frames are thus the first higher plant chloroplast genes identified as being indispensable.


Assuntos
DNA de Cloroplastos/genética , Genes Essenciais , Fases de Leitura Aberta/genética , Proteínas de Plantas/genética , Plantas Tóxicas , Tabaco/genética , Cloroplastos/genética , Mapeamento Cromossômico , Mutagênese Insercional , Plastídeos/genética , Polimorfismo de Fragmento de Restrição , Deleção de Sequência , Tabaco/citologia
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