RESUMO
Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.
Assuntos
Córtex Cerebelar/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial , Northern Blotting , Western Blotting , Divisão Celular , Córtex Cerebelar/crescimento & desenvolvimento , Primers do DNA , DNA Complementar/genética , Inibidores Enzimáticos/química , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares , Dados de Sequência Molecular , Peso Molecular , Morfogênese , Proteínas do Tecido Nervoso/química , Proteínas Nucleares , Células PC12/metabolismo , Fragmentos de Peptídeos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Precursores de Proteínas , Proteínas/química , Células de Purkinje/metabolismo , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas de Ligação a RNA , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sinapses/fisiologiaRESUMO
Differential display is a used widely and useful technique for the study of differentially expressed genes. However, very poor results have been obtained in the past when particular gene families were studied. Initially, we attempted to study the mRNA expression of catalytic subunits of serine/threonine phosphatases, using two primers specific to consensus sequences of these phosphatases. When differential display was applied, two wide, unresolved bands were isolated that contained cDNA of several phosphatases, together with that of many other unrelated transcripts. To overcome this problem, we used an alternative strategy, referred to as single strand differential display (SSDD), which is a combination of differential display and single strand conformation polymorphism (SSCP). After initial PCR amplification with specific primers, we ran a polyacrylamide (or agarose) gel, pre-selecting the region that contained fragments of the size expected for the consensus region (250-350 bp). The DNA eluted from this zone was then separated on a non-denaturing (SSCP) gel. Using this approach, we were able to characterize the expression of five ser/thr phosphatases, and a previously unreported splice variant of one of them, PP1gamma. All these phosphatases show varying levels of expression during development, indicating a very complex regulation of protein phosphorylation-dephosphorylation during the period of synaptogenesis in the mouse cerebellum.
Assuntos
Cerebelo/enzimologia , Cerebelo/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Fosfoproteínas Fosfatases/genética , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/análise , Processamento Alternativo/genética , Animais , Northern Blotting , Diferenciação Celular/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas Fosfatases/química , Fosforilação , Sinapses/genética , Sinapses/metabolismoRESUMO
1. Tau, which is a microtubule-associated protein, with mRNA targeted to the axon and growth cone, is involved in axonal elongation. During postnatal development in mouse, Tau expression in cerebellar granule cells is reduced afte the second postnatal week. The aim of this work was to study the regulation of the rate of the synthesis of Tau protein during the period of granule cell axonal growth in mouse cerebellum. 2. We found four [35S]methionine-labeled isoforms of Tau synthesized postnataly. Their levels remain constant from postnatal day 9 to 12 (P9-P12), and decreased by P20. 3. The rate of Tau synthesis showed differences with the rate of synthesis of total proteins. They also differ from proteins phosphatases 2A and 2B, both associated with the regulation of Tau function. In addition, the turnover of newly synthesized Tau increased at P20, compared with P9 and P12. 4. These results imply a specific developmental regulation of mRNA translation of Tau, and indicate that, after the period of synapse formation is complete, and therefore axonal growth has finished (P20), only a limited number of new Tau molecules are synthesized. This might reflect that, after synapse formation is complete, newly synthesized Tau molecules are not longer needed.
Assuntos
Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas tau/genética , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Calcineurina/biossíntese , Cerebelo/crescimento & desenvolvimento , Metionina/metabolismo , Camundongos , Fosfoproteínas Fosfatases/biossíntese , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Radioisótopos de Enxofre , Sinapses/fisiologia , Proteínas tau/biossínteseRESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first "synthetic antibody" reported.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Oligonucleotídeos/síntese química , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Imuno-Histoquímica , Camundongos , Oligonucleotídeos/imunologia , Oligonucleotídeos/isolamento & purificação , Reação em Cadeia da Polimerase , Testes de Precipitina , CoelhosRESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.
RESUMO
Oligonucleotide aptamers obtained using the SELEX procedure can recognize different molecules with high affinity. However, for proteins, this recognition is limited to native conformations and the specificity has not been clearly demonstrated by methods such as Western blotting, immunohistochemistry or immunoprecipitations. Using a library of oligonucleotides and a selection strategy based on high specificity instead of high affinity, we have reported previously the preparation of polyclonal oligobodies, reagents that recognize the protein PP2A in a very specific way. Here we report a method to obtain monoclonal oligobodies. The oligobody developed specifically recognized both native and denatured states of the protein CPD1 used as a model system. We further demonstrate the specificity of the monoclonal oligobody using Western blots, immunohistochemistry, and immunoprecipitation, procedures previously limited only to antibody-based detection. In addition, a confocal microscopy is shown that was obtained using an oligobody made by chemical synthesis using an oligonucleotide synthesizer, being this the first [quot ]synthetic antibody[quot ] reported.