Molecular Cloning And Characterization Of Dopamine Responsive Genes From Astrocytes

Molecular Cloning and Characterization of DopamineResponsive Genes From AstrocytesAbstractDopamine (3,4-dihydroxyphenethylamine; DA) is the predominant catecholamine neurotransmjtter in the mammalian central nervous system. DA controls a variety of functions including locomotor activity, cognition, emotion, positive reinforcement, food intake, and endocrine regulation. Of many slow-acting neurotransmitters, DA has received by far the most attention, mainly because that several pathological conditions have been linked to dysregulation of dopaminergic transmission. It has been found by a number of investigators that glial cells are targets of DA. However, few studies have reported on the molecular cascade associated with receptor activation and DA metabolism in glial cells during dopaminergic neuronal activity.In this study, a series of cellular and molecular biological technichies were applied to isolate and identify the DA responsive genes from glial cells.Analysis of DA responsive genes should provides a better understanding of the functional impact of glial cells in dopaminergic transmission, and the complex cellular and molecular mechanism of the disorder of dopaminergic transmission including Parkinson's disease, schizophrenia, and drug addiction etc.In the present study, we have investigated the influence of DA on gene transcription in primary glial cells. Two-directional (forward and backward) suppression subtraction hybridization (SSH) was performed on astrocytes primarily cultured from rat cerebral tissues in either standard media or treated with DA.PCR-select differential screening was used to further verifythe differentially expressed cDNA clones, and positive clones' weresequenced and the mRNAs were re-examined on Northern blots. Fourteensequences were identified, among which l1 were homologous to knowngenes, 3 were homologous to expressed sequence tags (ESTs). The analysisof all these identified sequences suggested that comp1ex intracellu1arsigna1ing, involving cross talks with growth factor pathway, steroidhormone pathway, and/or interferon-regulated 2-5A pathway, is induced byDA in astrocytes. The target genes of the signaling pathway were fOund tofall into fOur groups, including metabolic enzymes, stress proteins, transferproteins, and growth regulation proteins. In addition, several genes havebeen established their relationship with specific neurodegenerative diseases,showing that there is an over1ap in pathogenic mechanisms of thosediseases.Molecular cloning and functional analysis of human genes especialythose human disease genes are important biological tasks at present. DA isthe predominant catecholamine neurotransmitter in the mammalian centralnervous system, and several pathological conditions have been linked todysregulation of dopaminergic transmission. In the present study, we haveisolated a series of full-length cDNAs of DA responsive genes from huamnbrain using a homologous cloning strategy.A high-quality cDNA x phagelibrary with good representation of full-length cDNAs was constructed bySmart technique.Two runs of hybridization with SSH-PCR products of DAresonsive cDNA fragmnets (see Part one) as probe were performed, and 96positive clones were selected and sequenced, 6l candidate DA responsivegenes were isolated,of which 4 new full-length cDNAs were found andsubmitted to GeneBank.Analysis of these genes suggested that the complexmolecular cascade associated with receptor activation and DA metabolismin glial cells during dopaminergic neuronal activity and the complexIVcellular and molecular mechanism of the disorder of dopaminergictransmission.Some nove1 genes isolated laid a fOundation fOr furtherfunctional research and development of theraPy for dopaminergic diseases.Two important novel genes, a putative GTPase DRP and a LIMdomain transcription factor DATl, were selected fOr further studies basedon bioinfOrmatic ana1ysis. In this study, the rat full-length DRP a...