Degenerate Suppression PCR Identifies the ₂-Adrenergic Receptor as Upregulated by Neuronal Differentiation

Authors: LEWERENZ J.; LEYPOLDT F.; METHNER A.

Source: Gene Expression, Volume 11, Number 2, 2003 , pp. 105-116(12)

Publisher: Cognizant Communication Corporation

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Abstract:

Communication between cells is necessary for the functioning of a multicellular organism. Cells process a large amount of information through G-protein-coupled receptors, and activation of this receptor class has been implicated in neuronal differentiation. In this study, we used a method based on PCR with degenerated primers to identify G-protein-coupled receptors regulated by retinoic acid-induced differentiation of the human teratocarcinoma cell line NTera-2/D1. Subtracted cDNA libraries and control cDNA served as templates in half-sided PCR with a forward degenerate primer based on a conserved sequence from human serotonergic, adrenergic, and dopaminergic receptors and reverse primers on adaptors with long terminal repeats commonly employed in subtractive suppression hybridization. We developed conditions to amplify G-protein-coupled receptors from adaptor-ligated cDNA and found the bgr

₂-adrenergic receptor to be upregulated fourfold. This seems to be physiologically relevant, as it could also be shown in rat primary cortical cultures maturing in vitro. The method presented here makes use of the otherwise unused control cDNA from subtractive suppression hybridization experiments and could be easily adapted to other gene families.

Keywords: NT2 cells Neuronal differentiation bgr 2-Adrenergic receptor Subtractive suppression hybridization Degenerate PCR

Document Type: Research article

Affiliations: 1: Research Group Protective Signaling, Zentrum für Molekulare Neurobiologie and Department of Neurology, University Hospital Hamburg, Martinistr. 52, D-20246 Hamburg, Germany

Publication date: 2003-01-01

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The Molecular and Cellular Biology area of Gene Expression covers all aspects of the gene including it structure, functions, and regulation in prokaryotes, eukaryotes, and viruses; molecular and cell biological aspects of cell growth and development, chromatin structure and function. These include topics such as DNA replication, DNA repair, gene transcription, transcriptional control, RNA processing, posttranscriptional control, oncogenes, molecular mechanisms of action of hormones, molecular mechanism of cellular differentiation, growth and development, protein synthesis, and posttranslational control.
The Molecular and Cellular Neuroscience area of Gene Expression covers all aspects of gene expression as described but is devoted exclusively to the nervous system in health and disease. Topics include studies of neurogenesis, development, aging, and neurodegeneration. Complex neural systems, motor control, special senses, and higher cortical function, when viewed from the perspective of gene expression, are appropriate for the journal. Research related to molecular mechanisms of drug tolerance, dependence, and withdrawal are solicited. Manuscripts on state-of-the-art methods and protocols for molecular profiling of neuronal structure and function are welcome.