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
Our research objective was to characterize the biochemical effect of streptomycin during postnatal rat cerebral cortex development using a sensitive method that preserves the in situ topological relationship. We found a decrease in the mannosylation of asparagine-linked oligosaccharides without affecting polypeptide synthesis, DNA synthesis or glucose and mannose disappearance from the medium in mini-tissue units derived from P5. In addition, the rate of Dolp-GlcNAc2 Man9 Glc3 synthesis and the oligosaccharide protein transferase activity did not change in the presence of the aminoglycoside. These findings strongly suggested that the alteration of protein mannosylation occurred downstream of G3 transfer to nascent polypeptides. Further, the mini-tissue units may be useful for the assessment of neurological toxicity of antibacterial agents.
Assuntos
Córtex Cerebral/metabolismo , Hexosiltransferases , Proteínas de Membrana , Estreptomicina/farmacologia , Animais , Sequência de Carboidratos , Córtex Cerebral/efeitos dos fármacos , Glucose/metabolismo , Glicosilação/efeitos dos fármacos , Cinética , Manose/metabolismo , Dados de Sequência Molecular , Oligossacarídeos/biossíntese , Oligossacarídeos/química , Ratos , Ratos Wistar , Transferases/metabolismoRESUMO
Thy 1.2 is a well-known major cell surface glycoprotein of the central nervous system (CNS). However, the regulation of the expression of this molecule as well as its function are yet to be determined. To approach these problems we studied the synthesis of the molecule in the developing cerebellum of wild-type and staggerer mutant mice. We found the appearance of a [35S]-methionine-labeled band detected with specific Sepharose 4B-bound monoclonal antibodies (Mabs). The Thy 1.2 activity increases progressively from postnatal day 9 (P9), reaching the highest rate at P12, subsequently decreasing sharply at P13, and remaining relatively low up to P16 in the wild type. Comparison of these data to the rates of total protein synthesis reveals a selective developmental regulation of Thy 1.2 expression, at least at the translational level. This correlates quite well with the timing of synaptic stabilization between parallel fibers and Purkinje cell dendritic spines. Furthermore, at P12 Thy 1.2 protein is preferentially located in the synaptosomal fraction. The parallel fiber:Purkinje cell synapsis is not stabilized in the staggerer mutant mouse. At P12 Thy 1.2 synthesis is 30% of the wild type, indicating that the translational regulation of Thy 1.2 is altered in the staggerer mutation.