Identification of cis-Regulating Elements andtrans-Acting Factors Regulating the Expression of the Gene Encoding the Small Subunit of Ribonucleotide Reductase inDictyostelium discoideum*
- From the ‡Department of Biology, ¶Department of Chemistry and Biochemistry, and ‖Centre for Structural and Functional Genomics, Concordia University, Montreal, Quebec H3G 1M8, Canada
Abstract
We have examined the promoter ofrnrB, the gene encoding the small subunit of ribonucleotide reductase of Dictyostelium discoideum, usinglacZ 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 β-galactosidase. The behavior of these cis-regulatory elements implies that the mechanism regulating the prespore-specific expression ofrnrB 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.
Footnotes
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↵* This work was supported by National Sciences and Engineering Council (NSERC) of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Present address: MethylGene Inc., 7220 Frederick-Banting, Ville St-Laurent, Quebec H4S 2A1, Canada.
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↵** Recipient of an NSERC postgraduate scholarship.
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↵‡ To whom correspondence should be addressed: Dept. of Biology, Concordia University, 1455 de Maisonneuve Blvd. W., Montreal, Quebec H3G 1M8, Canada. Tel.: 514-848-3405; Fax: 514-848-2881; E-mail: tsang@vax2.concordia.ca.
- Abbreviations:
- CAE
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C/A-rich element
- PIPES
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1,4-piperazinediethanesulfonic acid
- PCR
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polymerase chain reaction
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- Received February 4, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
