RESUMO
The impact of the use of herbicides in agriculture can be minimized by compliance with good management practices that reduce the amount used and their release into the environment. Simple tests that provide real time on-site information about these chemicals are a major aid for these programs. In this work, we show that phage anti-immunocomplex assay (PHAIA), a method that uses phage-borne peptides to detect the formation of antibody-analyte immunocomplexes, is an advantageous technology to produce such field tests. A monoclonal antibody to the herbicide clomazone was raised and used in the development of conventional competitive and noncompetitive PHAIA immunoassays. The sensitivity attained with the PHAIA format was over 10 times higher than that of the competitive format. The cross-reactivity of the two methods was also compared using structurally related compounds, and we observed that the two-site binding of PHAIA "double-checks" the recognition of the analyte, thereby increasing the assay specificity. The positive readout of the noncompetitive PHAIA method allowed adaptation of the assay into a rapid and simple format where as little as 0.4 ng/mL clomazone (more than 10-fold lower than the proposed standard) in water samples from a rice field could be easily detected by simple visual inspection.
Assuntos
Herbicidas/análise , Imunoensaio/métodos , Isoxazóis/análise , Oxazolidinonas/análise , Biblioteca de Peptídeos , Poluentes Químicos da Água/análise , Animais , Anticorpos Monoclonais/imunologia , Bacteriófago M13/imunologia , Feminino , Herbicidas/imunologia , Isoxazóis/imunologia , Limite de Detecção , Camundongos , Camundongos Endogâmicos BALB C , Oxazolidinonas/imunologia , Poluentes Químicos da Água/imunologiaRESUMO
Enterococcus faecalis/faecium repeats (EFARs) are miniature insertion sequences spread in the genome of Enterococcus faecalis and Enterococcus faecium. Unit-length repeats measure 165-170 bp and contain two modules (B and T) capable of folding independently into stem-loop sequences, connected by a short, unstructured module J. The E. faecalis elements feature only one type of B, J and T modules. In contrast, the E. faecium elements result from the assembly of different types of B, J and T modules, and may vary in length because they carry multiple B modules. Most EFARs are located close (0-20 bp) to ORF stop codons, and are thus cotranscribed with upstream flanking genes. In both E. faecalis and E. faecium cells, EFAR transcripts accumulate in a strand-dependent fashion. Data suggest that T modules function as bidirectional transcriptional terminators, which provide a 3'-end to gene transcripts spanning B modules, while blocking antisense transcripts coming in from the opposite direction.
Assuntos
DNA Bacteriano/genética , Enterococcus faecalis/genética , Enterococcus faecium/genética , Sequências Repetidas Invertidas , Sequência de Bases , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
BACKGROUND: All bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA). RESULTS: Stenotrophomonas maltophilia is an environmental bacterium increasingly involved in nosocomial infections and resistant to most antibiotics. The availability of the whole DNA sequence of the S. maltophilia strain K279a allowed us to set up fast and accurate PCR-based diagnostic protocols based on the measurement of length variations of loci carrying a variable number of short palindromic repeats marking the S. maltophilia genome. On the basis of the amplimers size, it was possible to deduce the number of repeats present at 12 different loci in a collection of S. maltophilia isolates, and therefore label each of them with a digit. PCR-negative regions were labelled 0. Co-amplification of two pairs of loci provided a 4-digit code sufficient for immediate subtyping. By increasing the number of loci analyzed, it should be possible to assign a more specific digit profile to isolates. In general, MLVA data match genotyping data obtained by PFGE (pulsed-field gel electrophoresis). However, some isolates exhibiting the same PCR profiles at all loci display distinct PFGE patterns. CONCLUSION: The utilization of the present protocol allows to type several S. maltophilia isolates in hours. The results are immediately interpretable without the need for sophisticated softwares. The data can be easily reproducible, and compared among different laboratories.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Reação em Cadeia da Polimerase/métodos , Stenotrophomonas maltophilia/classificação , Stenotrophomonas maltophilia/genética , Eletroforese em Gel de Campo Pulsado , Genoma Bacteriano/genética , Genótipo , Sequências Repetitivas de Ácido Nucleico/genética , Stenotrophomonas maltophilia/isolamento & purificaçãoRESUMO
Pro-inflammatory cytokines, environmental stresses, as well as receptor tyrosine kinases regulate the activity of JNK. In turn, JNK phosphorylates Jun members of the AP-1 family of transcription factors, thereby controlling processes as different as cell growth, differentiation, and apoptosis. Still, very few targets of the JNK-Jun pathway have been identified. Here we show that JNK is required for the induction of c-myc expression by PDGF. Furthermore, we identify a phylogenetically conserved AP-1-responsive element in the promoter of the c-myc proto-oncogene that recruits in vivo the c-Jun and JunD AP-1 family members and controls the PDGF-dependent transactivation of the c-myc promoter. These findings suggest the existence of a novel biochemical route linking tyrosine kinase receptors, such as those for PDGF, and c-myc expression through JNK activation of AP-1 transcription factors. They also provide a novel potential mechanism by which both JNK and Jun proteins may exert either their proliferative or apoptotic potential by stimulating the expression of the c-myc proto-oncogene.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Animais , Apoptose , Sequência de Bases , Northern Blotting , Western Blotting , Divisão Celular , Cromatina/metabolismo , Drosophila , Ativação Enzimática , Genes Reporter , Humanos , MAP Quinase Quinase 4 , Sistema de Sinalização das MAP Quinases , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Células NIH 3T3 , Filogenia , Testes de Precipitina , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Ativação Transcricional , TransfecçãoRESUMO
The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret oncogene, carrying either the cysteine 634 to tyrosine or the methionine 918 to threonine substitutions. These mutations convert the receptor tyrosine kinase, Ret, into a dominant transforming protein and induce constitutive activation of its intrinsic tyrosine kinase activity leading to congenital and sporadic cancers in neuroendocrine organs. Using the PC12, rat pheochromocytoma cell line, as model system, we show that Shp-2 mediates immediate-early gene expression if induced by either of the mutant alleles. Furthermore, we show that Shp-2 activity is required for RetM918T-induced Akt activation. The results indicate that Shp-2 is a downstream mediator of the mutated receptors RetC634Y and RetM918T, thus suggesting that it may act as a limiting factor in Ret-associated endocrine tumors, in the neoplastic syndromes multiple endocrine neoplasia types 2A and 2B.
Assuntos
Membranas Intracelulares/fisiologia , Mutação/fisiologia , Proteínas Oncogênicas/genética , Proteínas Tirosina Fosfatases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Peptídeos e Proteínas de Sinalização Intracelular , Fatores de Crescimento Neural/metabolismo , Proteínas Oncogênicas/metabolismo , Células PC12/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
Neisseria miniature insertion sequences (nemis) are miniature DNA insertion sequences found in Neisseria species. Out of 57 elements closely flanking cellular genes analyzed by PCR, most were conserved in Neisseria meningitidis but not in N. lactamica strains. Since mRNAs spanning nemis are processed by RNase III at hairpins formed by element termini, gene sets could selectively be regulated in meningococci at the posttranscriptional level.
Assuntos
Elementos de DNA Transponíveis , DNA Bacteriano/química , Neisseria/genética , Sequências Repetitivas de Ácido Nucleico , Fases de Leitura Aberta , Reação em Cadeia da PolimeraseRESUMO
Approx. 2% of the Neisseria meningitidis genome consists of small DNA insertion sequences known as Correia or nemis elements, which feature TIRs (terminal inverted repeats) of 26-27 bp in length. Elements interspersed with coding regions are co-transcribed with flanking genes into mRNAs, processed at double-stranded RNA structures formed by TIRs. N. meningitidis RNase III (endoribonuclease III) is sufficient to process nemis+ RNAs. RNA hairpins formed by nemis with the same termini (26/26 and 27/27 repeats) are cleaved. By contrast, bulged hairpins formed by 26/27 repeats inhibit cleavage, both in vitro and in vivo. In electrophoretic mobility shift assays, all hairpin types formed similar retarded complexes upon incubation with RNase III. The levels of corresponding nemis+ and nemis- mRNAs, and the relative stabilities of RNA segments processed from nemis+ transcripts in vitro, may both vary significantly.
Assuntos
Endorribonucleases/química , Neisseria meningitidis/genética , Processamento Pós-Transcricional do RNA , RNA Bacteriano/química , RNA Mensageiro/química , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fases de Leitura Aberta/genética , Estabilidade de RNA/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Ribonuclease III , Elementos Nucleotídeos Curtos e Dispersos , Sequências Repetidas TerminaisRESUMO
In bacteria, conditions that uncouple translation from transcription activate intragenic terminators located within cistrons. We analyzed the function of NusA in intragenic termination, making use of two tandem terminators located within the hisG cistron, GTTE1 and GTTE2. GTTE2 is a canonical Rho site, capable to terminate with Rho alone in vitro. By contrast, GTTE1 is a suboptimal terminator, featuring a boxA element and requiring a functional NusB to terminate efficiently in vivo. We found that a functional NusA is necessary for efficient termination events to occur at both GTTE1 and 2. To enhance termination at GTTE1 in conditions in which the transcript is free of ribosomes, NusA acts at the same step as NusB and NusE/S10. In the presence of concomitant translation, termination at GTTE1 is dependent on the relative position of the translation stop codon and boxA. If translation stops upstream of boxA, NusA acts at the same step as NusB enhance termination. Ribosomes terminating translation at boxA influence termination at GTTE1. Interactions of NusA and/or NusB with ribosomal components, including NusE/S10, might facilitate termination. Differently from what observed at GTTE1, the NusA-stimulated pausing seems to be sufficient for the occurrence of complete termination events at GTTE2. A functional NusA is also necessary to prevent premature termination of normally translated transcripts. Our data support the hypothesis that NusA may program a fraction of the RNA polymerase to terminate transcription upon interactions with specific sites on the nascent mRNA and either other Nuses or ribosomes.
Assuntos
Proteínas de Escherichia coli , Fatores de Alongamento de Peptídeos/fisiologia , Biossíntese de Proteínas/genética , Regiões Terminadoras Genéticas/genética , Fatores de Transcrição/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Sequência de Bases , Sítios de Ligação/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Histidina/biossíntese , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Óperon/genética , Fatores de Alongamento de Peptídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica/genética , Fatores de Elongação da TranscriçãoRESUMO
About 2% of the Neisseria meningitidis genome is made up by nemis, short DNA sequences which feature long terminal inverted repeats (TIRs). Most nemis are interspersed with single-copy DNA and are found at close distance from cellular genes. In this work, we demonstrate than RNAs spanning nemis of different length and sequence compositions are specifically cleaved at hairpins formed by nemis termini by total cellular lysates derived from both Escherichia coli and Neisseria lactamica strains. The use of cellular extracts from E. coli strains impaired in the activity of known ribonucleases let to establish that cleavage at nemis TIRs is specifically mediated by the endoribonuclease RNase III. Data set the base for the identification of all of the neisserial genes that are regulated by RNase III because of their physical association with nemis DNA.
Assuntos
Proteínas de Escherichia coli , Neisseria meningitidis/genética , Sítios de Ligação , Endorribonucleases/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Neisseria meningitidis/enzimologia , Plasmídeos , Ribonuclease III , Especificidade da Espécie , Sequências Repetidas TerminaisRESUMO
Interactions between the antiterminator NusB and boxA elements in the nut sites are necessary to ensure lambda N-mediated processive antitermination. Similarly, in the bacterial cell, interactions between NusB and boxA elements help RNA polymerase to counteract polarity during transcription of rrn operons. We analyzed the effects of NusB on intragenic termination at the level of two tandem terminators located in the hisG cistron, GTTE1 and GTTE2. Unexpectedly, we found that NusB enhances transcription termination at the sub-optimal Rho site GTTE1. Moreover, site-directed mutagenesis of a boxA homolog located within GTTE1 and the masking of this element by translating ribosomes demonstrated that the recruitment of NusB in the termination complex is mediated by a boxA element. The mutated boxA also abolishes the formation of a NusB-dependent complex on GTTE1 RNA. On the whole, results provide evidence that interactions between NusB and boxA can enhance Rho-dependent termination.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Salmonella typhimurium/genética , Regiões Terminadoras Genéticas/genética , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Mutação/genética , Óperon/genética , Biossíntese de Proteínas , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Ribossomos/metabolismo , Sequências de Repetição em Tandem/genética , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
The association between the nutritional state of mongrel dogs naturally infected with Trypanosoma cruzi and their infectivity to Triatoma infestans bugs and immune response to Trypanosoma cruzi were studied in the rural village of Amamá, northwestern Argentina. All of the 97 evaluated dogs were classified into one of three categories of external clinical aspect (ECA) based on the degree of muscle development, external evidence of bone structures, state of the hair of the coat, existence of fatty deposits, and facial expression. ECA was significantly associated with two nutritional indicators, hematocrit and skin-fold thickness, but not with total serum proteins. For all dogs, hematocrit was significantly correlated with skin-fold thickness. The 2-year survival probability decreased significantly from 60.7% for dogs with good ECA to 45.9% and 31.2% for those with regular and bad ECA, respectively. The age-adjusted relative odds of infection for Triatoma infestans xeno-diagnosis nymphs that fed once on a dog seroreactive for Trypanosoma cruzi decreased significantly as ECA improved, when tested by multiple logistic regression analysis. A delayed hypersensitivity reaction was observed in all of the seroreactive dogs with good ECA but only in 45-50% of those with regular or bad ECA. Dogs with bad ECA had a 2.6 and 6.3 times greater probability of infecting triatomines after a single full blood meal than dogs with regular or good ECA, respectively. Our study shows that the reservoir competence of dogs for Trypanosoma cruzi was associated with ECA, which is a surrogate and valid index of nutritional state.
Assuntos
Doença de Chagas/veterinária , Doenças do Cão/parasitologia , Ectoparasitoses/veterinária , Distúrbios Nutricionais/veterinária , Animais , Anticorpos Antiprotozoários/sangue , Argentina/epidemiologia , Proteínas Sanguíneas/análise , Doença de Chagas/sangue , Doença de Chagas/complicações , Doença de Chagas/mortalidade , Doença de Chagas/transmissão , Estudos Transversais , Doenças do Cão/mortalidade , Cães , Ectoparasitoses/complicações , Ectoparasitoses/epidemiologia , Ectoparasitoses/transmissão , Feminino , Hematócrito/veterinária , Insetos Vetores/parasitologia , Masculino , Distúrbios Nutricionais/complicações , Distúrbios Nutricionais/mortalidade , Distúrbios Nutricionais/parasitologia , Estado Nutricional , Dobras Cutâneas , Taxa de Sobrevida , Triatoma/parasitologia , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/patogenicidadeRESUMO
Two subfractions with opposite immunological properties were obtained from the flagellar antigens (FF) of Trypanosoma cruzi epimastigotes by immunoaffinity chromatography. The ligand-bound material (Ag 123) contained four polypeptide bands of 97, 55, 38 and 14 kDa. The nonretained flow-through (FT), induced a potent proliferation of murine naive splenocytes. In contrast, Ag 123 inhibited the proliferative capacity of the FT as well as the proliferation mediated by the mitogen Concanavalin A (Con A). The suppressive effect of Ag 123 on the Con A-mediated proliferation was neutralized by an anti-TGF-beta monoclonal antibody. Both Ag 123 and FF stimulated high serum levels of TGF-beta in injected mice. Ag 123 also induced in vitro secretion of TGF-beta by murine splenocytes. These results demonstrate that Ag 123 is a potent stimulator of TGF-beta both in vivo and in vitro. Oligopeptides derived from the 38 kDa protein present in Ag 123 showed homology with human and rat alpha-fetoproteins (AFP). Ag 123 seems to have a key role in the immunosuppression that develops during early stages in the infection with T. cruzi.
Assuntos
Antígenos de Protozoários/farmacologia , Cromatografia de Afinidade/métodos , Ativação Linfocitária/efeitos dos fármacos , Fator de Crescimento Transformador beta/fisiologia , Trypanosoma cruzi/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Protozoários/análise , Concanavalina A/farmacologia , Eletroforese em Gel de Poliacrilamida , Feminino , Flagelos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeo Hidrolases , Peptídeos/análise , Baço/citologia , Frações Subcelulares/imunologia , Fator de Crescimento Transformador beta/efeitos dos fármacos , Fator de Crescimento Transformador beta/imunologiaRESUMO
Activity-based costing is a method of calculating cost of a service, focusing on operations. It gives quick and tangible cost information to operations and financial managers. While this method has be used more in the manufacturing area, it is gaining acceptance in the medical practice. This article describes activity-based costing and illustrates how to start utilizing it in a practice.
Assuntos
Alocação de Custos/métodos , Administração Financeira/métodos , Prática Privada/economia , Coleta de Dados , Renda , Sistemas de Informação Administrativa , Modelos Organizacionais , Ortopedia/economia , Prática Privada/organização & administração , Tennessee , Estados Unidos , Revisão da Utilização de Recursos de SaúdeRESUMO
The flagellar fraction (FF) of Trypanosoma cruzi can be separated by immunoaffinity chromatography in two fractions with balanced but opposite immunological effects. The immunoaffinity purified fraction has immunosuppressive activity mediated at least partially by TGF-beta (Hansen et al., submitted). Here we report that the fraction depleted of immunosuppresive antigens (FT) administered with iscom-matrix as adjuvant provides enhanced protection to an infection challenge in immunized mice. In vitro, the FT but not the FF stimulated resident peritoneal cells to produce IL-1 and IL-6. In immunized mice, the FT elicited higher levels of antigen-specific IgG2a than the FF as well as broader recognition of T. cruzi antigens. Splenocytes from mice immunized with FT proliferated spontaneously in vitro and secreted TH1 and TH2 cytokines. The protection provided by FT correlates with its capacity to enhance the secretion of IFN-gamma. We postulate that immunosuppressive antigens present in the FF prevent the development of memory cells secreting IFN-gamma through a TGF-beta dependent mechanism.
Assuntos
Antígenos de Protozoários/administração & dosagem , Flagelos/imunologia , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Doença de Chagas/imunologia , Doença de Chagas/prevenção & controle , Citocinas/biossíntese , Imunização , Imunoglobulina G/sangue , Memória Imunológica , Imunossupressores/isolamento & purificação , Técnicas In Vitro , Interferon gama/biossíntese , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The polycistronic mRNA of the histidine operon is subject to a processing event that generates a rather stable transcript encompassing the five distal cistrons. The molecular mechanisms by which such a transcript is produced were investigated in Escherichia coli strains carrying mutations in several genes for exo- and endonucleases. The experimental approach made use of S1 nuclease protection assays on in vivo synthesized transcripts, site-directed mutagenesis and construction of chimeric plasmids, dissection of the processing reaction by RNA mobility retardation experiments, and in vitro RNA degradation assays with cellular extracts. We have found that processing requires (1) a functional endonuclease E; (2) target site(s) for this activity in the RNA region upstream of the 5' end of the processed transcript that can be substituted by another well-characterized rne-dependent cleavage site; (3) efficient translation initiation of the first cistron immediately downstream of the 5' end; and (4) a functional endonuclease P that seems to act on the processing products generated by ribonuclease E. This is the first evidence that ribonuclease P, an essential ribozyme required for the biosynthesis of tRNA, may also be involved in the segmental stabilization of a mRNA.
Assuntos
Endorribonucleases/metabolismo , Proteínas de Escherichia coli , Genes Bacterianos , Complexos Multienzimáticos , Óperon , Processamento Pós-Transcricional do RNA , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genótipo , Histidina/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Mapeamento por Restrição , Ribonuclease P , Ribossomos/metabolismo , Transaminases/biossíntese , Transaminases/genética , Transcrição GênicaRESUMO
Previous studies have shown that the expression of the his operon of Salmonella typhimurium is regulated at the level of transcription initiation, transcription elongation and RNA processing. We have analyzed his RNA in both prototrophic strains or strains harboring regulatory and auxotrophic mutations grown under a variety of metabolic conditions that lead to differential expression of the operon. Under some of these conditions, there is an increase in the amount of prematurely released his-specific RNA, resulting in modulation of the relative amount of full-length transcripts. Under the same metabolic conditions, there is also a modulation of RNA processing events that generate a very stable RNA species comprising the five distal cistrons. These effects appear to be due to perturbation of the translation process caused by alterations in the intracellular pool of initiator transfer RNA.
Assuntos
Regulação Bacteriana da Expressão Gênica , Histidina/genética , Óperon , Salmonella typhi/genética , Transcrição Gênica , RNA MensageiroRESUMO
In vivo analysis of expression of the chloroplast rDNA cluster during somatic embryogenesis of Daucus carota (D.carota) was performed by Northern-blot analysis with different DNA probes, spanning both the 16S rRNA gene, the 16S-23S rRNA spacer, which contains the two mosaic tRNA genes tRNA(Ile) and tRNA(Ala), and the region upstream of the 16S rRNA gene, where a tRNA(Val) maps. We show that expression both of the spacer tRNAs tRNA(Ile) and tRNA(Ala) is not significantly regulated during development whereas the amount of the transcript corresponding to tRNA(Val) is not detectable during early embryonic stages and progressively accumulates during late phases. Multiple transcription start sites have been identified upstream of the tRNA(Val) gene by S1 mapping analysis, which are activated late during the embryogenesis. These data indicate that developmental control mechanisms act on plastid gene expression during embryogenesis in carrot.
Assuntos
Cloroplastos/metabolismo , DNA Ribossômico/genética , RNA de Transferência/genética , Verduras/genética , Sequência de Bases , Northern Blotting , Células Cultivadas , Mapeamento Cromossômico , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Regulação da Expressão Gênica , Dados de Sequência Molecular , RNA de Transferência de Alanina/genética , RNA de Transferência de Isoleucina/genética , RNA de Transferência de Valina/genética , Transcrição Gênica , Verduras/crescimento & desenvolvimentoRESUMO
Post-transcriptional mechanisms operate in regulation of gene expression in bacteria, the amount of a given gene product being also dependent on the inactivation rate of its own message. Moreover, segmental differences in mRNA stability of polycistronic transcripts may be responsible for differential expression of genes clustered in operons. Given the absence of 5' to 3' exoribonucleolytic activities in prokaryotes, both endoribonucleases and 3' to 5' exoribonucleases are involved in chemical decay of mRNA. As the 3' to 5' exoribonucleolytic activities are readily blocked by stem-loop structures which are usual at the 3' ends of bacterial messages, the rate of decay is primarily determined by the rate of the first endonucleolytic cleavage within the transcripts, after which the resulting mRNA intermediates are degraded by the 3' to 5' exoribonucleases. Consequently, the stability of a given transcript is determined by the accessibility of suitable target sites to endonucleolytic activities. A considerable number of bacterial messages decay with a net 5' to 3' directionality. Two different alternative models have been proposed to explain such a finding, the first invoking the presence of functional coupling between degradation and the movement of the ribosomes along the transcripts, the second one implying the existence of a 5' to 3' processive '5' binding nuclease'. The different systems by which these two current models of mRNA decay have been tested will be presented with particular emphasis on polycistronic transcripts.
Assuntos
Bactérias/genética , Modelos Genéticos , Processamento Pós-Transcricional do RNA/fisiologia , RNA Mensageiro/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genéticaRESUMO
A 3457-base pair fragment of Azospirillum brasilense DNA which complemented mutations in the hisA and hisF genes of Escherichia coli was sequenced. The sequence analysis revealed the presence of six major contiguous open reading frames (ORF). The comparison of the predicted amino acid sequence of these ORF with those encoded by the eubacterial, archaebacterial and eukaryotic his genes sequenced thus far revealed that four of them have a significant degree of homology with the E. coli hisH, hisA, hisF and the C-terminal domain of the hisI gene products. S1 mapping experiments indicated that the putative transcription start site coincided with the AUG translational initiation codon of the hisBd gene, the first gene of the A. brasilense his operon. Downstream from the last ORF, a sequence was identified which functions as a Rho-independent transcription terminator. Comparison of amino acid sequences, gene order and organization and evolutionary aspects of the A. brasilense his cluster are discussed.