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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA004154-02
Application #
3209381
Study Section
Pharmacology I Research Subcommittee (DABR)
Project Start
1985-09-01
Project End
1987-11-30
Budget Start
1986-09-01
Budget End
1987-11-30
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Type
Overall Medical
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Couceyro, P; Douglass, J (1995) Precipitated morphine withdrawal stimulates multiple activator protein-1 signaling pathways in rat brain. Mol Pharmacol 47:29-39
Douglass, J; McKinzie, A A; Couceyro, P (1995) PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine. J Neurosci 15:2471-81
Douglass, J; McKinzie, A A; Pollock, K M (1994) Identification of multiple DNA elements regulating basal and protein kinase A-induced transcriptional expression of the rat prodynorphin gene. Mol Endocrinol 8:333-44
Couceyro, P; Pollock, K M; Drews, K et al. (1994) Cocaine differentially regulates activator protein-1 mRNA levels and DNA-binding complexes in the rat striatum and cerebellum. Mol Pharmacol 46:667-76
Cai, Y C; Douglass, J (1993) In vivo and in vitro phosphorylation of the T lymphocyte type n (Kv1.3) potassium channel. J Biol Chem 268:23720-7
McMurray, C T; Pollock, K M; Douglass, J (1992) Cellular and molecular analysis of opioid peptide gene expression. NIDA Res Monogr 126:113-31
Kaynard, A H; McMurray, C T; Douglass, J et al. (1992) Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity. Mol Endocrinol 6:2244-56
Cai, Y C; Osborne, P B; North, R A et al. (1992) Characterization and functional expression of genomic DNA encoding the human lymphocyte type n potassium channel. DNA Cell Biol 11:163-72
Douglass, J O; Christie, M; Adelman, J P et al. (1991) Characterization of mammalian potassium channel genes. NIDA Res Monogr 111:54-68
Douglass, J; Grimes, L; Shook, J et al. (1991) Systemic administration of kainic acid differentially regulates the levels of prodynorphin and proenkephalin mRNA and peptides in the rat hippocampus. Brain Res Mol Brain Res 9:79-86

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