RESUMO
Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter-lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity.
Assuntos
Inibidores da Captação de Dopamina/toxicidade , Dopamina/análise , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Perfilação da Expressão Gênica , Biblioteca Gênica , Metanfetamina/toxicidade , Proteínas do Tecido Nervoso/biossíntese , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ácido 3,4-Di-Hidroxifenilacético/análise , Animais , DNA Complementar/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Indução Enzimática , Feminino , Genes Sintéticos , Óperon Lac , Masculino , Mesencéfalo/citologia , Mesencéfalo/embriologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Ratos , Transcrição Gênica , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.
Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Placenta/metabolismo , Proteínas da Gravidez/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , DNA Complementar , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Gravidez , Proteínas da Gravidez/química , Homologia de Sequência de AminoácidosRESUMO
Targeted sequencing of the mouse t-complex has started with a 176-kb, gene-rich BAC localized with six PCR-based markers in inversion 2/3 of the highly duplicated region. The sequence contains 11 genes recovered primarily as cDNAs from early embryonic collections, including Igfals (previously placed on chromosome 17), Nubp2 (a fully characterized gene), Jsap1 (a JNK-binding protein), Rsp29 (the mouse homologue of the rat gene), Ndk3 (a nucleoside diphosphate kinase), and six additional putative genes of unknown function. With 50% GC content, 75% of the DNA transcribed, and one gene/16.0 kb (on average), the region may qualify as one of the most gene-dense segments in the mouse genome and provides candidates for dosage-sensitive phenotypes and mouse embryonic lethals mapped to the vicinity.
Assuntos
DNA/genética , Família Multigênica/genética , Animais , Proteínas de Transporte/genética , Cromossomos Bacterianos/genética , Ilhas de CpG/genética , DNA Complementar/genética , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica de Plantas , Glicoproteínas/genética , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Núcleosídeo-Difosfato Quinase/genética , Isoformas de Proteínas/genética , Ratos , Homologia de Sequência do Ácido Nucleico , Tioléster Hidrolases/genéticaRESUMO
Little is known about gene action in the preimplantation events that initiate mammalian development. Based on cDNA collections made from each stage from egg to blastocyst, 25438 3'-ESTs were derived, and represent 9718 genes, half of them novel. Thus, a considerable fraction of mammalian genes is dedicated to embryonic expression. This study reveals profound changes in gene expression that include the transient induction of transcripts at each stage. These results raise the possibility that development is driven by the action of a series of stage-specific expressed genes. The new genes, 798 of them placed on the mouse genetic map, provide entry points for analyses of human and mouse developmental disorders.
