RESUMO
A method is described that allows cDNA production from individual brain cell clones or 'neurospheres'. These culture-generated spheres of stem, progenitor, and differentiated cells have been the focus of interest because they represent an in vitro model of neurogenesis. However, because neurospheres are somewhat resistant, in part due to their enclosure by a dense extracellular matrix, to methods attempting to disrupt them and isolate nucleic acids, there is a need for new technology that affords the simple and efficient RT-PCR for studies of neural gene expression and discovery. A method is described here that uses sonication and an all-in-one approach for the construction of cDNA from single neurospheres. The generation of cDNA from individual adult brain stem/progenitor cell neurospheres is useful for future studies of neurogenic gene expression.
Assuntos
Encéfalo/citologia , Técnicas de Cultura de Células/métodos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células-Tronco/fisiologia , Animais , Células Clonais , Primers do DNA , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/fisiologia , Sonicação , Células-Tronco/citologiaRESUMO
Two recombinant bacteriophage lambda clones encoding a 27.8-kb fragment of the human phosphodiesterase beta-subunit gene were isolated from a human genomic library. The nucleotide sequences of 19 exons (from the 4th to 22nd), 18 introns, and the 3'-flanking region were determined. The analysis of the nucleotide sequence of the phosphodiesterase beta-subunit gene revealed four Alu repeats and four minisatellite regions.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Diester Fosfórico Hidrolases , Células Fotorreceptoras/enzimologia , Sequência de Aminoácidos , Bacteriófago lambda/genética , Sequência de Bases , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , DNA Complementar , DNA Satélite , Escherichia coli/genética , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Retina/enzimologiaRESUMO
Guanylate kinase (EC 2.7.4.8), catalysing the reaction GMP+ATP = GDP+ADP, was purified to homogeneity from bovine retina. Primary structure of the enzyme was determined by parallel analyses of amino acid sequences of its peptides and nucleotide sequence of the corresponding cDNA. It is shown that the bovine retinal guanylate kinase like the analogous enzyme from yeast Saccharomyces cerevisiae contains a characteristic glycine-rich motif, involved in ATP binding. All of the amino acids, involved in GMP binding in the yeast enzyme, are conserved or conservatively substituted in the bovine retinal guanylate kinase. The bovine retinal enzyme was expressed in E. coli as a fusion protein. Data are presented on the purification of the fusion protein, its digestion by enteropeptidase, purification of the recombinant enzyme and its functional characteristics.
Assuntos
Guanilato Ciclase/isolamento & purificação , Retina/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Guanilato Ciclase/química , Guanilato Ciclase/genética , Dados de Sequência Molecular , Plasmídeos , Homologia de Sequência de AminoácidosRESUMO
Guanylate kinase (EC 2.7.4.8) catalyzing the reaction GMP + ATP = GDP + ADP, was purified to homogeneity from bovine retina. Using oligonucleotides based on the amino acid sequence of this enzyme, the cDNA encoding guanylate kinase (GK) was isolated and its nucleotide sequence was determined. Expression of the GK cDNA in E. coli, and the purification and functional characterization of the expressed enzyme are presented. It is shown that bovine retinal GK, like its yeast counterpart, contains the characteristic glycine-rich motif and all the amino acids involved in GMP binding. Bovine retinal enzyme is extended for several amino acid residues both at the N- and C-termini, compared to the yeast enzyme.
Assuntos
Clonagem Molecular , GMP Cíclico/metabolismo , Expressão Gênica , Núcleosídeo-Fosfato Quinase/genética , Retina/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA Complementar/química , DNA Complementar/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Guanilato Quinases , Ligação de Hidrogênio , Dados de Sequência Molecular , Núcleosídeo-Fosfato Quinase/isolamento & purificação , Plasmídeos , Proteínas Recombinantes de Fusão , Análise de Sequência , Homologia de Sequência , Transformação Bacteriana , Tripsina/metabolismoRESUMO
The primary structure of the bovine retinal calcium binding protein P26 has been determined by the parallel analysis of the protein and the corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. P26 was expressed in E. coli as a fusion protein and, after purification by affinity chromatography on IgG-Sepharose 6, cleaved off with enteropeptidase.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Escherichia coli/genética , Proteínas do Olho/genética , Cristalino/metabolismo , Células Fotorreceptoras/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Bovinos , DNA , Eletroforese em Gel de Poliacrilamida , Proteínas do Olho/metabolismo , Expressão Gênica , Dados de Sequência Molecular , Plasmídeos , Recoverina , Relação Estrutura-AtividadeRESUMO
The primary structure of bovine retinal calcium binding protein P26 has been determined by parallel analysis of protein and corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. Preliminary data are presented on expression of P26 as a fusion protein in E. coli.