Inducible expression of exogenous genes in Dictyostelium discoideum using the ribonucleotide reductase promoter.
We report here the development of a regulated gene expression system for Dictyostelium discoideum based on the DNA-damage inducibility of the rnrB gene. rnrB, which codes for the small subunit of the enzyme ribonucleotide reductase, responds to DNA-damaging agents at all stages of the D.discoideum life cycle. Doses that have little effect on development have previously been shown to increase the level of the rnrB transcript by up to 15-fold. Here we show that all elements necessary for DNA-damage induction are contained in a 450 bp promoter fragment. We used a fusion of the rnrB promoter with the gene encoding GFP to demonstrate an up to 10-fold induction at the RNA level, which appears in all aspects similar to induction of the endogenous rnrB transcript. Using a fusion with the lacZ gene we observed an up to 7-fold induction at the protein level. These results indicate that the rnrB promoter can be used to regulate the expression of specific genes in D.discoideum. This controllable gene expression system provides the following new characteristics: the induction is rapid, taking place in the order of minutes, and the promoter is responsive at all stages of the D.discoideum life cycle.
Gaudet
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P
Department of Chemistry and Biochemistry, Centre for Structural and Functional Genomics, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec H3G 1M8, Canada.
MacWilliams
H
H
Tsang
A
A
D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology; D000818:Animals; D004249:DNA Damage; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000138:chemical synthesis; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000096:biosynthesis / Q000235:genetics*; D014466:Ultraviolet Rays; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D008164:Luminescent Proteins; D009153:Mutagens; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015112:4-Nitroquinoline-1-oxide; D008741:Methyl Methanesulfonate; D012264:Ribonucleotide Reductases; D001616:beta-Galactosidase