In the past two or three years it has been shown that there are at least three different genes involved in the production of opioids. One gene codes for pro-opiomelanocortin (POMC) from which Beta-endorphin, ACTH and melanocyte stimulating hormones are derived. A second gene codes for pro-enkephalin which contains multiple copies of leu-and met-enkephalin and other enkephalin peptides. A third gene codes for prodynorphin, the common precursor to Alpha-neo-endorphin and dynorphin. The major long range goal of the proposed research is to better understand how the expression of each of the opioid peptide genes is regulated in the pituitary, brain and other sites in the body. We will use cDNA probes isolated from cDNA clone banks to identify and sequence the genes and determine their chromosomal locations. Specific genomic DNA probes will then be used to study how expression of the genes is regulated at the levels of transcription, and metabolism of the mRNA using intact animals, cell lines, and primary cell culture systems. Genomic DNA probes isolated from lambda genomic libraries will be made to specific regions of each gene to determine if the genes undergo any structural change (i.e. methylation) or rearrangements following teatment with various agents, or during the onset of pathological processes that affect expression of the genes. These molecular hybridization probes will also be used to determine where each class of peptide is synthesized in laboratory animals and humans by measuring mRNA levels in various tissues. In addition, we will determine the effect of various regulators and drugs such as morphine and steriods on the mRNA levels in each tissue and in different neuronal pathways. Finally, transfer of genes and segments of genes from expressing cell types to non-expressing cell types (and vice versa) will be performed to delineate those portions of the genes and their flanking sequences that are required for expression. Gene transfer experiments will also be performed to define specific amino acid sequences in the precursor proteins that are necessary for correct proteolytic processing of these polyproteins.
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