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J Biol Chem, Vol. 274, Issue 29, 20384-20390, July 16, 1999

Identification of cis-Regulating Elements and trans-Acting Factors Regulating the Expression of the Gene Encoding the Small Subunit of Ribonucleotide Reductase in Dictyostelium discoideum

Claire Bonfils, Pascale Gaudet^¶, and Adrian Tsang^¶

From the  Department of Biology, ^¶ Department of Chemistry and Biochemistry, and  Centre for Structural and Functional Genomics, Concordia University, Montreal, Quebec H3G 1M8, Canada

We have examined the promoter of rnrB, the gene encoding the small subunit of ribonucleotide reductase of Dictyostelium discoideum, using lacZ as a reporter gene. Deletion analysis showed that expression of this gene in vegetative cells involves an A/T-rich element, whereas its expression in prespore cells during development requires a region encompassing two G/C-rich elements, designated box A and box B. Removal of boxes A and B results in very low level of activity. When either box A or box B is deleted, prestalk cells adjacent to the prespore zone also express -galactosidase. The behavior of these cis-regulatory elements implies that the mechanism regulating the prespore-specific expression of rnrB is different from that regulating other known prespore genes.

We have used electrophoretic mobility shift assays to identify factors that interact with box A and box B. Box A interacts with a factor that is found in the nuclear fraction. While box B interacts with a factor that is present in the cytosolic fraction throughout growth and development, its presence in the nuclear fraction is developmentally regulated. Results from competition assays suggest that both box A and box B interact with transcriptional activators that have not been characterized previously.

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