The prodynorphin precursor molecule encodes the dynorphin family of opioid peptides. The prodynorphin gene is transcriptionally active in numerous CNS tissues and cell types, where the dynorphin opioid peptides regulate a variety of critically important physiological and behavioral responses such as pain perception, feeding, sleep, motor function, intestinal peristalsis and perhaps narcotic tolerance. When G-protein coupled membrane receptors on neurons expressing the prodynorphin gene are activated by their target neurotransmitter, numerous cellular processes are altered. This general process is referred to as neural plasticity, and includes the phenomenon of trans-synaptic regulation of transcription. In order to understand the molecular events which mediate trans-synaptic regulation of transcription it is necessary to characterize the chemical messengers which transfer information from the cell membrane to a specific genomic locus, as well as the specific protein factors and their target nucleotide sequences which regulate transcription in response to those signals. The major goals of the research proposed here are aimed at characterizing the nuclear events underlying trans-synaptic regulation of prodynorphin gene expression. Molecular biological approaches will be used to accomplish three specific aims: 1) Northern blot and RNase analysis will define the sites of synthesis and structure of human and primate prodynorphin mRNA. Gene transfer approaches will define the human prodynorphin promoter region. This work will lay the foundation for future studies regarding regulated expression of the human prodynorphin gene; 2) Gene transfer protocols will define the promoter region(s) of the rat prodynorphin gene, with deletion analysis defining nucleotide sequences regulating transcription control by various second messengers. A novel gene regulatory affinity binding system will be used to scan over 3.5kb of sequenced rat prodynorphin 5' flanking DNA for the presence of DNA binding proteins; 3) We will define the nucleotide sequence which is responsible for the binding of a neuronal-specific DNA binding protein, Dyn1. This protein is observed exclusively in CNS tissues, and may be responsible for the controlled expression of genes in neuronal cell types. Once the nucleic acid binding site is determined, schemes will be employed to further characterize the factor using protein purification or expression cloning protocols.
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