The cloning, expression and partial characterization of a human delta opioid receptor.

Persistent Link:
http://hdl.handle.net/10150/186784
Title:
The cloning, expression and partial characterization of a human delta opioid receptor.
Author:
Fang, Lei.
Issue Date:
1994
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
A human delta (δ) opioid receptor (hDOR) was cloned from human brain cDNA libraries by filter replica hybridization screening methods. A single clone (44-11) from a human striatal cDNA library was obtained with a 467 bp cDNA probe produced by PCR amplification using NG 108-15 cells cDNA as the template. The 44-11 clone contained a 0.7 kb fragment corresponding to the 3' end of the mouse δ opioid receptor (mDOR) open reading frame (ORF) including the TGA stop codon. The 0.7 kb segment derived from the 44-11 clone by EcoR I and Not I restriction enzyme digestion, was used to screen a human temporal cortical cDNA library. Three positive 1.0 kb clones were obtained from this library. None of the clones contained the full length δ opioid receptor ORF. Sequence analysis showed that two of these clones were identical to each other and also identical to a portion of the 44-11 clone. However, one of these clones 78-4, was shown to contain a 0.64 kb fragment corresponding to the 5' end of the mDOR ORF including the ATG start codon. Since the 44-11 and 78-4 clones shared identical overlapping regions and a unique Hinc II restriction site, a human cDNA containing a full length of the δ opioid receptor ORF was reconstructed by ligation of these two clones at the Hinc II site. The reassembled human cDNA (78x44) has the full length human δ opioid receptor (hDOR) ORF. It contains 1116 nucleotides encoding a protein of 372 amino acids with 93% amino acid identity to the mDOR and the rat δ opioid receptor (rDOR). The 78x44 cDNA was subcloned into the pcDNA3 expression vector and transiently transfected into COS-7 cells. These cells expressed 1.0 pmole of [³H]naltrindole binding/mg protein. Delta opioid receptor selective ligands exhibited high affinity at the receptors while μ and κ selective ligands showed low affinity at the receptors. These results show that the expressed protein in COS-7 cells transfected with 78x44-pcDNA3 is the human δ opioid receptor.
Type:
text; Dissertation-Reproduction (electronic)
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Pharmacology and Toxicology; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Yamamura, Henry I.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleThe cloning, expression and partial characterization of a human delta opioid receptor.en_US
dc.creatorFang, Lei.en_US
dc.contributor.authorFang, Lei.en_US
dc.date.issued1994en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractA human delta (δ) opioid receptor (hDOR) was cloned from human brain cDNA libraries by filter replica hybridization screening methods. A single clone (44-11) from a human striatal cDNA library was obtained with a 467 bp cDNA probe produced by PCR amplification using NG 108-15 cells cDNA as the template. The 44-11 clone contained a 0.7 kb fragment corresponding to the 3' end of the mouse δ opioid receptor (mDOR) open reading frame (ORF) including the TGA stop codon. The 0.7 kb segment derived from the 44-11 clone by EcoR I and Not I restriction enzyme digestion, was used to screen a human temporal cortical cDNA library. Three positive 1.0 kb clones were obtained from this library. None of the clones contained the full length δ opioid receptor ORF. Sequence analysis showed that two of these clones were identical to each other and also identical to a portion of the 44-11 clone. However, one of these clones 78-4, was shown to contain a 0.64 kb fragment corresponding to the 5' end of the mDOR ORF including the ATG start codon. Since the 44-11 and 78-4 clones shared identical overlapping regions and a unique Hinc II restriction site, a human cDNA containing a full length of the δ opioid receptor ORF was reconstructed by ligation of these two clones at the Hinc II site. The reassembled human cDNA (78x44) has the full length human δ opioid receptor (hDOR) ORF. It contains 1116 nucleotides encoding a protein of 372 amino acids with 93% amino acid identity to the mDOR and the rat δ opioid receptor (rDOR). The 78x44 cDNA was subcloned into the pcDNA3 expression vector and transiently transfected into COS-7 cells. These cells expressed 1.0 pmole of [³H]naltrindole binding/mg protein. Delta opioid receptor selective ligands exhibited high affinity at the receptors while μ and κ selective ligands showed low affinity at the receptors. These results show that the expressed protein in COS-7 cells transfected with 78x44-pcDNA3 is the human δ opioid receptor.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmacology and Toxicologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairYamamura, Henry I.en_US
dc.contributor.committeememberRoeske, William R.en_US
dc.contributor.committeememberPorreca, Franken_US
dc.contributor.committeememberDavis, Thomas P.en_US
dc.contributor.committeememberLaird, Hugh E.en_US
dc.identifier.proquest9432851en_US
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