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Identifier uch.biology.msc//2003gkouskou
Title Λειτουργική συσχέτιση της πρωτεΐνης Rad9 με τον χαλκορυθμιζόμενο μεταγραφικό παράγοντα Mac1 στον Saccharomyces cerevisiae
Creator Gkouskou, Kalliopi
Abstract In Saccharomyces cerevisiae, Mac1 is a copper-regulated DNA binding transcription factor that, under copper depletion conditions, activates genes involved in copper uptake (CTR1, CTR3, FRE1, FRE7). Previous evidence suggested that, in addition to copper, protein interactions could modulate Mac1 function (Voutsina et al, 2000). In order to identify such proteins, a genomic expression library was screened using the two hybrid technology (A. Voutsina unpublished data). Among the potentially Mac1-interacting proteins, Rad9 was identified, a nuclear phosphoprotein, a prototype DNA-damage checkpoint protein required for the DNA checkpoint pathway through out the cell cycle. In this study there has been an effort to confirm the interaction between Rad9 and Mac1, to examine the physiological conditions under which the interaction takes place, and to initiate an analysis of the possible biological role of this interaction. In vivo coimmunoprecipitation assays of Rad9 and Mac1 showed that the two proteins interact in extracts that were taken from cells grown either in media depleted from copper or containing low copper. Besides that, domains of Rad9 were subcloned in appropriate expression vectors to identify in vitro the specific region of Rad9 that interacts with Mac1. Moreover, the accumulation of CTR1 mRNA in compared to wild type cultures grown under high copper, low copper or copper starvation was measured. We found CTR1 transcription induced in rad9Δ cells in the presence of high or low copper. Therefore, Rad9 may act directly or indirectly as a repressor of CTR1 transcription. We also measured the transcriptional activity of LexAMac1, tethered to LexAbinding DNA sites, in rad9Δ compared to wild type cells to see if the transactivation function of Mac1 was affected. We observed that Mac1 transcriptional activity was reduced in cells grown in low copper. These initial results, although not easily explained, are quite challenging to further investigate the physiological role of Mac1-Rad9 interaction.
Issue date 2003-11-01
Date available 2003-12-10
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work--Post-graduate theses
Permanent Link https://elocus.lib.uoc.gr//dlib/d/4/5/metadata-dlib-2003gkouskou.tkl Bookmark and Share
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