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Regulation of the ribonucleotide reductase small subunit gene in Dictyostelium discoideum

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Regulation of the ribonucleotide reductase small subunit gene in Dictyostelium discoideum

Gaudet, Pascale (2001) Regulation of the ribonucleotide reductase small subunit gene in Dictyostelium discoideum. PhD thesis, Concordia University.

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Abstract

Ribonucleotide reductase catalyzes the reduction of ribonucleotides to deoxyribonucleotides, providing precursors for the synthesis of DNA. Expression of ribonucleotide reductase is correlated with DNA synthesis: it is up-regulated during the DNA synthesis phase of the cell cycle and in the course of DNA repair. We have examined the regulation of expression of the ribonucleotide reductase small subunit gene of Dictyostelium discoideum, rnrB , during the cell cycle, in response to DNA-damaging agents and during development. Our results suggest that rnrB is expressed during two periods of the cell cycle in Dictyostelium , with one expression peak in mid-G2 and one in late G2. A cis -acting element referred to as box A appears to be able to confer cell-cycle-regulated expression. We have shown that the level of rnrB transcript increases when cells are treated with mutagens and with hydroxyurea, an inhibitor of ribonucleotide reductase. The response is rapid, transient and independent of protein synthesis. A DNA fragment consisting of the 450 bp upstream of the start codon of rnrB has been shown to be sufficient to confer DNA-damage inducibility on heterologous genes. We have used deletion analysis to define the cis -acting elements of the rnrB promoter required for the response to two different DNA-damaging agents, methyl methane sulfonate and 4-nitroquinoline-1-oxide. Our results indicate that box C can confer response to both drugs, while box A and box D confer response to methyl methane sulfonate and 4-nitroquinoline-1-oxide, respectively. We have studied the phenotype of a mutant in which part of the rnrB promoter has been deleted by gene replacement. The mutant strain fails to up-regulate the rnrB gene in response to DNA-damaging agents. This mutant displays increased sensitivity to mutagens as well as prolonged cell cycle arrest upon exposure to mutagens. Our laboratory has shown by histochemical staining that the rnrB gene is expressed only in the posterior, prespore zone during development. We have identified by deletion analysis and site-directed mutagenesis cis-acting elements responsible for cell-type-specific expression of rnrB during development. Preventing the expression of rnrB does not appear to cause morphological defects in Dictyostelium development. Using electrophoretic mobility shift assays, we have detected cellular factors that may regulate the expression of the rnrB gene

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Gaudet, Pascale
Pagination:xv, 116 leaves : ill. (some col.) ; 29 cm.
Institution:Concordia University
Degree Name:Theses (Ph.D.)
Program:Chemistry and Biochemistry
Date:2001
Thesis Supervisor(s):Tsang, Adrian S
ID Code:1321
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:18
Last Modified:08 Dec 2010 10:19
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