Date of Award

8-1-1996

Document Type

Thesis

Degree Name

Master of Arts (MA)

Department

Biology

First Advisor

Dr. Bruce Chase

Abstract

The biogenic amine octopamine is a versatile signaling molecule that acts in essential physiological functions in invertebrates. Its roles in neurotransmission, neurohormone regulation and neuromodulation are achieved through interaction with G-protein coupled receptors. Such invertebrate receptors are structurally and functionally analogous to vertebrate adrenergic receptors. A human brain β2-adrenergic receptor cDNA clone was used as a hybridization probe to identify a Drosophila gene encoding an octopamine/tyramine receptor. This 367 base pair fragment of Drosophila genomic DNA, homologous to the putative sixth and seventh transmembrane regions of adrenergic receptors, was used to isolate an octopamine/tyramine receptor 3.3 Kb cDNA clone from a Drosophila head cDNA library. A 2.2 Kb fragment of this cDNA encoding the octopamine/tyramine receptor protein was used as a hybridization probe to obtain several lambda clones containing Drosophila genomic DNA spanning the receptor’s gene. This thesis presents experiments that address the structure of this gene. It describes the subcloning and analysis of three specific genomic fragments. A 1.0 Kb EcoRI genomic DNA fragment and a 1.3 Kb HindIII/EcoRI genomic DNA fragment were isolated from a Drosophila genomic phage (#1). A 3.4 Kb HindIII/EcoR1 genomic DNA fragment was also isolated from a Drosophila genomic phage (#10). DNA sequence information from the 1.0 Kb genomic DNA fragment and the 1.3 Kb genomic DNA fragment that were subcloned show that they contain sequence that is homologous to the 5’ end of the 3.3 Kb protein coding region cDNA relative to the restriction map of the genomic DNA. This has allowed the establishment of the 5’ to 3’ orientation of the transcript at the gene and also has allowed inferences as to the approximate location of the promoter and poly-A sites. Analysis of restriction mapping and sequence data of the 1.0 Kb genomic DNA fragment and analysis of the 1.3 Kb genomic DNA fragment that are homologous to the 5’ end of the 2.2 Kb protein encoding region cDNA of the octopamine/tyramine receptor also provided evidence for the existence of at least two introns in the 5’ coding region of this genes primary mRNA. The 3.4 Kb genomic DNA fragment shows homology to the 2.2 Kb protein coding region of the 3.3 Kb cDNA of the octopamine/tyramine receptor gene in Southern blot analysis, but shows no sequence homology at either its 5’ or 3’ ends. Thus it too may contain intronic sequence.

Comments

A Thesis Presented to the Department of Biology and the Faculty of the Graduate College University of Nebraska In Partial Fulfillment of the Requirements for the Degree Master of Arts University of Nebraska at Omaha. Copyright 1996 Kelley Lynn Colvin.

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