Untersuchung der Nutzung unterschiedlicher Transkriptionsstartpunkte für mRNA von humanem Kathepsin D unter dem Einfluß von Calcitriol

In der vorliegenden Arbeit wurden die Transkriptionsstartstellen für mRNA von humanem Kathepsin D unter dem Einfluß von Calcitriol in U937-Zellen untersucht. Zu diesem Zweck wurde die Methode der kompetitiven RT-PCR der Fragestellung entsprechend modifiziert. Die Methode der kompetitiven PCR basie...

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Bibliographic Details
Main Author: Wagenknecht, Christian Martin
Contributors: Hasilik, Andrej (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2006
Online Access:PDF Full Text
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In the presented paper, the transcription start sites of mRNA from human cathepsin D were examined under the influence of calcitriol in U937 cells. Therefor, the method of the competitive RT-PCR was modified according to the problem. The method of the competitive PCR is based on co-amplification of a specific target-sequence (DNA resp. reverse transcripted RNA) - i.e. the amount of DNA that has to be determined - together with known amounts of an internal standard (competitor, standard-DNA) in the same reaction tube. The method allows the detection of even smallest amounts of DNA resp. RNA and at the same time their quantification. The therefor needed competitor could be developed through in vitro mutagenesis, except from a deletion of 30 bp, it is identical with the sequence to be determined. There was isolated and examined mRNA from calcitriol-stimulated U937 cells and from control U937 cells. The results in the paper show, that in both cases several transcription start sites for cathepsin D are used. Furthermore it could be shown an approximately 8-fold enhancement of the transcription rate for cathepsin D mRNA under stimulation with calcitriol. It could not be demonstrated, that a main use of a single start site is the reason for the enhanced transcription rate, like it is for example in estrogen stimulated MCF7 cells.