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Identification of Genes Regulated by Pro-Leu-Gly-NH2 (PLG) Using Differential Display RT-PCR (ddPCR).

Costain, WJ (Department of Psychiatry and Behavioural Neuroscience, McMaster University, Canada)
Mishra, RK (Department of Psychiatry and Behavioural Neuroscience, McMaster University, Canada)

Contact Person: Willard J Costain (costainw@fhs.mcmaster.ca)


PLG is an endogenously derived hypothalamic factor originally described as melanocyte stimulating hormone release inhibiting factor (MIF-1). Soon after, the ability of PLG to regulate central dopaminergic neurotransmission was discovered. Of particular interest to us is the manner in which PLG exerts an effect on the function of dopamine D2-receptors in the basal ganglia. In vivo studies have shown that PLG potentiates the behavioral effects of dopamine agonists (enhanced rotation in the hemi-Parkinson's rat and enhanced arousal in Parkinsonian patients), attenuates neuroleptic and morphine induced catalepsy and dopamine receptor supersensitivity, antagonizes oxotremorine induced tremor, potentiates CPP (NMDA receptor antagonist) induced locomotion and darting and protects against MPTP induced degeneration of nigrostriatal dopaminergic neurons. In vitro radioligand binding studies have shown that PLG increases the binding of D2 agonists in striatal membranes, modulates the high affinity state of D2 receptors and attenuates neuroleptic-induced D2 receptor supersensitivity. The ability of PLG to modulate the function of central dopaminergic pathways indicates a potential therapeutic benefit in the treatment of Parkinson's and schizophrenia. We have screened approximately 2400 genes using ddPCR (RNAimage® kit1, GeneHunter Corp.) and have identified 3 bands which are regulated by treatment with PLG (20 mg/kg i.p. for 21 days). Each of the three bands were originally identified as being down regulated (compared to control) as a result of PLG treatment. The down regulated bands were excised from the 6% polyacrylamide sequencing gels and reamplified by PCR using the same primers with which they were generated. The reamplified PCR products were cloned into the pCR-Trap® cloning system (GeneHunter Corp.). Insertion of cDNA was confirmed by colony PCR. Sequencing of the instered cDNAs (dideoxy chain-termination method, Mobix facility, McMaster University) revealed a total of 6 unique cDNA species. Hind III restriction digestion of the plasmid DNA was seperated on a 2% agarose gel and the inserted cDNA was recovered using the QIAEX II method. The cDNA were recoved from the agarose gel (QIAEX II, QIAGEN Corp.) and labelled using random primed labelling kit (Gibco) for use as a probe in a northern blot. The sequence information for the insert cDNA was obtained (Mobix facility, McMaster University) and comparison with GenBank revealed no significant sequence homologies. Confirmation of the cDNA expression was done using Northern Hybridization. Blots were stripped and reprobed with b-actin for accurate determination of expression levels.

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Poster Number PAcostain0764
Keywords: neuropeptide, striatum, rat, ddPCR, dopamine

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Costain, WJ; Mishra, RK; (1998). Identification of Genes Regulated by Pro-Leu-Gly-NH2 (PLG) Using Differential Display RT-PCR (ddPCR).. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Available at URL http://www.mcmaster.ca/inabis98/neuroscience/costain0764/index.html
© 1998 Author(s) Hold Copyright