RESUMO
A 3-year-old castrated male domestic short-haired cat was presented with nodules on the left nasal wing and the left earflap. Prototheca cells were found after excision biopsy of one of the nodules located on the left earflap. The patient cat was generally in good condition without skin problems. Prototheca wickerhamii was isolated from all 6 masses after they were surgically nucleated. The cat was recovered two months after intervention with no recurrence of skin nodules. This report deals with the first case of feline protothecosis in Japan.
Assuntos
Doenças do Gato/patologia , Doenças do Gato/cirurgia , Prototheca/isolamento & purificação , Prototheca/patogenicidade , Animais , Biópsia , Gatos , Masculino , Orquiectomia , Prototheca/crescimento & desenvolvimentoRESUMO
The nucleotide sequence surrounding the translation initiation AUG codon (AUG context) is important for the effective translation initiation. A compilation analysis revealed that all the genes of the eukaryotic translation initiation factor 1, which plays a crucial role in the recognition of the optimal AUG context, ironically have extremely bad AUG contexts.
Assuntos
Códon de Iniciação , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Biossíntese de Proteínas , Animais , Sequência de Bases , Humanos , RNA Mensageiro/químicaRESUMO
The implementation of efficient technologies for the production of recombinant mammalian membrane receptors is an outstanding challenge in understanding receptor-ligand actions and the development of therapeutic antibodies. In order to improve the solubility of recombinant extracellular domains of human membrane receptors expressed in Escherichia coli, proteins were synthesized by an E. coli in vitro translation system supplemented with bacterial molecular chaperones, such as GroEL-GroES (GroEL/ES), Trigger factor (TF), a DnaK-DnaJ-GrpE chaperone system (DnaKJE), and/or a heat shock protein Hsp100, ClpB. The following three proteins that are prone to aggregation were examined: the extracellular domain (ECD) or the second immunoglobulin-like domain (IgII) of the human neurotrophin receptor TrkC (TrkC-ECD and TrkC-IgII), and the C-type lectin carbohydrate recognition domain of the human asialoglycoprotein receptor (ASGPR HI CRD). The cooperative chaperone system including GroEL/ES, DnaKJE and ClpB had a marked effect on the solubility of TrkC-ECD and TrkC-IgII, and the GroEL/ES-DnaKJE-TF chaperone system was more effective for TrkC-IgII. The GroEL/ES-DnaKJE-TF chaperone network increased the yield of soluble ASGPR HI CRD. The present findings demonstrate that E. coli molecular chaperones are useful in improving the yield of soluble recombinant extracellular domains of human membrane receptors in an E. coli expression system.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli , Chaperonas Moleculares/metabolismo , Receptores de Superfície Celular/biossíntese , Proteínas Recombinantes/biossíntese , Sistema Livre de Células/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Chaperonas Moleculares/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/genética , SolubilidadeRESUMO
The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.
Assuntos
Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Ribulose-Bifosfato Carboxilase/biossíntese , Ribulose-Bifosfato Carboxilase/genética , Synechococcus/genética , Escherichia coli/genética , Mutação da Fase de Leitura/genética , Plasmídeos/genética , Biossíntese de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Synechococcus/enzimologiaRESUMO
The CO(2)-regulatory function of the AT-rich element in the promoter for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbc) genes in the cyanobacterium Synechococcus sp. PCC7002 was analyzed using the transcription factor decoy approach. Double-stranded phosphorothioate AT-rich oligonucleotides with high affinity for a sequence-specific DNA-binding protein were successfully introduced into cyanobacterial cells in culture without any transfection reagent. The AT-rich decoy oligonucleotides interfered with CO(2) regulation of rbc expression by blocking the binding of the sequence-specific DNA-binding protein, indicating that the AT-rich element plays a critical role in CO(2) regulation for rbc genes. The decoy oligonucleotide approach to cyanobacteria provides a simple and excellent tool for investigating transcriptional regulation in vivo.
Assuntos
Dióxido de Carbono/antagonistas & inibidores , Cianobactérias/enzimologia , Regulação Bacteriana da Expressão Gênica , Oligonucleotídeos/farmacologia , Ribulose-Bifosfato Carboxilase/genética , Sequência Rica em At , Transporte Biológico , Cianobactérias/efeitos dos fármacos , Cianobactérias/genética , Proteínas de Ligação a DNA/metabolismo , Repressão Enzimática , Fluoresceína-5-Isotiocianato , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Ativação Transcricional/efeitos dos fármacosRESUMO
We analyzed the promoter of the genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (rbc) in the cyanobacterium Synechococcus sp. PCC7002 and localized the CO(2)-regulatory element. Cyanobacterial transformants were constructed with several DNA segments of the rbc promoter fused to the chloramphenicol acetyltransferase (CAT) gene, and their acetyltransferase activities were analyzed under 0.03% and 1% CO(2) conditions. We found that the AT-rich element localized from -262 to -291 relative to the rbc translation-starting site was required for CO(2)-dependent repression. Fluorescent-labeled oligonucleotide probes of identical sequence to the AT-rich element were reacted with protein extracts from cells cultured under conditions of low and high CO(2) atmospheric content. We detected a gel retardation complex of a strong signal intensity in extracts from cells cultured under 15% CO(2), but only a weak signal from cells cultured under 1% CO(2). Moreover, a DNA affinity precipitation assay identified a 16-kDa protein that bound to nucleotide sequences within the AT-rich element. The partial amino acid sequence of the protein was similar to the deduced protein sequences of ORF129 and ORF155 from Synechocystis 6803. Our findings suggest that the AT-rich element plays a role as a negative CO(2)-regulatory element and its trans-acting factor possibly regulates the rbc transcription in response to CO(2) levels.
Assuntos
Dióxido de Carbono/farmacologia , Cianobactérias/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Ribulose-Bifosfato Carboxilase/genética , Transativadores/metabolismo , Adenosina Trifosfatases/efeitos dos fármacos , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Cianobactérias/genética , Cianobactérias/metabolismo , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Ribulose-Bifosfato Carboxilase/metabolismo , Transativadores/genéticaRESUMO
We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay proved reliable measurement and multiple detection of DNA binding proteins.
