RESUMO
Nitric oxide (NO) has a profound role in the generation, differentiation, survival, and physiology of neurons. We have created a novel transgenic model to study the action of NO in the adult brain, in which the neuronal isoform of NO synthase (nNOS) is expressed under control of the promoter of the calcium-calmodulin multifunctional kinase IIalpha (CaMKIIalpha) gene. We show that the transgenic nNOS RNA and protein are expressed in the cortex, the hippocampus and the striatum of the transgenic mice. We also show that expression of several genes involved in the protection of neurons from oxidative stress and cell death is not affected in neurons of the transgenic mice. Furthermore, generation of new cells is depressed in the neurogenic brain areas in transgenics. In addition, we analyze gene expression in the hippocampus of the transgenic animals using microarray RNA profiling and Q-PCR. Our experiments describe specific changes in cell division and gene activity in the CaMKII-nNOS transgenic model and demonstrate its utility for studying the action of NO in the adult brain.
Assuntos
Regulação Enzimológica da Expressão Gênica , Sistema Nervoso/metabolismo , Óxido Nítrico Sintase Tipo I/biossíntese , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular/genética , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Perfilação da Expressão Gênica , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Neurônios/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA/biossíntese , RNA/genética , TransgenesRESUMO
Conjugation in the freshwater ciliate Tetrahymena thermophila involves a developmental program that models meiosis, fertilization, and early developmental events characteristic of multicellular eukaryotes. We describe a gallery of five early-acting conjugation mutations. These mutants, cnj1-5, exhibit phenotypes in which specific steps in the conjugal pathway have been altered or eliminated. Specifically, cnj1 and cnj2 fail to condense their micronuclear chromatin prior to each of the three prezygotic nuclear divisions. This results in nuclear division failure, failure to replicate DNA, and failure to initiate postzygotic development. The cnj3 mutant appears to exhibit a defect in chromosome separation during anaphase of mitosis. cnj4 mutants successfully carry out meiosis I, yet are unable to execute the second meiotic division and abort all further development. cnj5 mutants are unable to initiate either meiosis I or meiosis II, yet proceed to execute all subsequent developmental events. These mutant phenotypes are used to draw inferences regarding developmental dependencies that exist within the conjugation program.