Disruption of the ard expression, encoding a subunit of Drosophila neuronal nicotinic acetylcholine receptor by mitomycin C-oligonucleotide construct and ribozyme.
Item
-
Title
-
Disruption of the ard expression, encoding a subunit of Drosophila neuronal nicotinic acetylcholine receptor by mitomycin C-oligonucleotide construct and ribozyme.
-
Identifier
-
AAI3169963
-
identifier
-
3169963
-
Creator
-
Pierzchala, Marek.
-
Contributor
-
Adviser: Thomas Schmidt-Glenewinkel
-
Date
-
2005
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Chemistry, Biochemistry | Biology, Molecular
-
Abstract
-
The goal of this studies it to disrupt the expression of the and gene encoding one of the subunits of the nAChR in Drosophila. The results may lead to better understanding of the role the ARD subunit plays and the function and structure of the nicotinic receptor. Out of many approaches to influence the expression of the ard gene we had chosen two direct methods; first---using MC-oligonucleotide conjugate---we attempted to induce small mutation in crucial stretch of ard DNA sequence, and second-using ribozyme---we tried to reduce or abolish ard mRNA, thus preventing or reducing efficient translation. Mitomycin C is able to alkylate and cross-link DNA strands; it is also a potent mutagen. MC-oligonucleotide constructs were designed and synthesized. The cross-linking was done in vitro on M13mp18 vector. M13mp18 vector from the cross-linking reaction was transformed into E. coli to investigate the nature of the potential mutations. Also, cross-linking that could lead to a mutation was attempted in vivo in E. coli. In addition, similar attempt was tried in Drosophila by injecting MC-oligonucleotide constructs into thousands of Drosophila embryos. In spite of laborious screenings potential mutations were not identified.;Another method to disrupt efficient gene expression is through destruction of mRNA by employing targeted ribozymes. We used several ribozymes against ard mRNA. DNA coding for the ribozymes was subcloned into pP{lcub}CaSpeR-hs) vector. Using P-element mediated transformation several transgenic Drosophila lines were established. Drosophila embryos from each line were heat-shocked to allow the subcloned ribozyme DNA, that was under the control of heat-shock promoter, to be transcribed into the specific ribozyme and cleave the and mRNA. Immunoblotting experiments indicate that the ARD protein level, in the larvae hatched form the heat-shocked embryos, was substantially decreased, presumably by the action of the hairpin ribozyme.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
