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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

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ribonucleotide reductase catalyzes the conversion of ribonucleotides to deoxyribonucleotides. As well, it is involved in maintaining a balanced pool of deoxyribonucleotides required for DNA synthesis (reviewed in Ref. 1). The central role played by ribonucleotide reductase in DNA synthesis has made it an important target for the design of anti-tumor and anti-viral drugs (reviewed in Ref. 2). In eukaryotes, this enzyme comprises two large and two small subunits (3). The expression of ribonucleotide reductase is periodic and is coordinated with DNA synthesis in a variety of organisms characterized. An increased level of the small subunit is detected during the S phase of the cell cycle (4-7) and during DNA repair (8-12). The expression of ribonucleotide reductase appears to be tightly regulated at the transcriptional and posttranscriptional levels. A thorough understanding of the mechanisms involved in its regulation is expected to shed additional insights into the control of DNA synthesis and DNA repair and potentially lead to rational drug design.

Supplied with ample nutrients, cells of the cellular slime mold Dictyostelium discoideum multiply by binary fission as single-celled amoebae. Depletion of food triggers the onset of a 24-h developmental program. The cells come together to form multicellular aggregates 8 h after the initiation of development. By 16 h, the multicellular aggregates called slugs are differentiated along the anterior-posterior axis. Prestalk cells occupy the anterior one-quarter of the slug, and prespore cells are located in the remaining three-quarters. These precursor cells ultimately differentiate into stalk cells and spores of the mature fruiting body. Prior to differentiation, cells in the prespore region undergo a wave of DNA synthesis (13-17). The role of this developmentally programmed burst of DNA synthesis is unknown. It has been suggested by different investigators to fuel cell division (13-15), mitochondrial replication (16), or both (17). Temporally and spatially correlated with this wave of DNA synthesis is the elevated expression of the gene encoding the small subunit of ribonucleotide reductase, rnrB (18). As in other organisms, it appears that fluctuations in the expression of rnrB can be used to predict changes in the rates of DNA synthesis. Altering the pattern of rnrB expression may be used as a tool to change the profile of DNA synthesis in evaluating the role of DNA synthesis in development.

Manipulating the regulatory regions of promoters provides a convenient way of changing the pattern of gene expression. The regulatory regions of several genes that are expressed predominantly in prespore cells have been characterized. Most of these promoters contain consensus C/A-rich elements (CAEs),¹ which have been shown to be important for transcriptional activity (19-23). Also required is an A/T-rich element located downstream of the CAEs (19, 20). When joined with a heterologous basal promoter, neither the CAEs nor the A/T-rich element alone is able to drive expression in prespore cells. However, expression in prespore cells can be stimulated when the CAEs and the A/T-rich element are placed together with a heterologous basal promoter (19, 20). The CAEs exhibit strong affinity for the developmentally regulated transcriptional factor GBF (24). Cells carrying a null mutation in the gene encoding GBF are arrested at the loose aggregate stage, before cell differentiation has occurred (24). The latter results provide further evidence that besides the interaction between GBF and CAEs, prespore gene expression requires the interaction with other factors and regulatory elements.

The regulation of rnrB appears to be more complex than that of the other known prespore genes. In addition to expression in prespore cells, it is expressed in vegetative cells. A cursory examination of the G/C-rich sequences in the promoter region of rnrB shows the absence of known cis-acting elements. Only one G/C-rich sequence in the promoter of rnrB exhibits similarity to half of a C/A-rich element. Here we show by deletion analysis that expression of rnrB in vegetative cells does not require any of the G/C-rich sequences found in the promoter. In addition, we have identified two G/C-rich sequences that can direct prespore expression during postaggregative development. Results from electrophoretic mobility shift experiments suggest that these two G/C-rich sequences interact with factors that are distinct from the transcriptional factor GBF.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth, Development, and Transformation of D. discoideum Cells-- Cells of the axenic strain AX2 were grown either axenically in HL5 medium (25) or on lawns of Enterobacter aerogenes on SM agar (26). At the logarithmic phase, 2-4 × 10⁶ cells/ml in HL5 or when the bacterial lawns began to clear, the cells were harvested and washed in ice-cold KKP buffer (20 mM KH₂PO₄/K₂HPO₄, pH 6.2) and allowed to develop on a solid substratum as we have previously described (27).

Plasmid DNAs from deletion constructs were introduced into AX2 cells by calcium phosphate coprecipitation as described previously (28). Transformants were selected in HL5 containing 20 µg/ml G418 (Life Technologies, Inc.). Over 50 independent transformants were pooled for analysis.

Primer Extension-- The oligonucleotide 5'-GAGAATTGGTTCAATGAATG-3', complementary to positions 26-45 of the sense strand of rnrB, was end-labeled with T4 polynucleotide kinase (Roche Molecular Biochemicals) and used as a primer. Approximately 10 fmol of the labeled oligonucleotide, 2 × 10³ cpm, were annealed at 41 °C for 16 h to 4 µg of poly(A)⁺ RNA in a reaction volume of 10 µl containing 10 mM PIPES, pH 6.4, and 0.4 M NaCl. After annealing, the RNA was transferred to a tube containing the extension reaction mix (50 mM Tris·Cl, pH 7.6, 10 mM MgCl₂, 1 mM dithiothreitol, 0.5 mM of each dNTP, 2 µg of actinomycin D, and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega) in a final volume of 100 µl) and incubated at 37 °C for 1 h. The radiolabeled extension product was then resolved alongside a sequencing ladder on a 6% polyacrylamide sequencing gel.

Construction of Promoter Deletions-- The XbaI/BamHI genomic fragment (29) contains two-thirds of the coding region and 450 base pairs of 5'-noncoding region of rnrB. This fragment was cloned in frame to lacZ into the XbaI/BglII sites of pDdGal16 (30) to generate construct 450. To construct the other 5' deletions, sequences were progressively removed with Bal31 (31) from the XbaI site. The cleaved DNA fragments were excised with BamHI and cloned into the BglII site and the end-filled HindIII site of pDdGal16. The end points of the deletions, marked in Fig. 1B, were determined by sequencing using the primer described for primer extension. With the exception of 450, all of the 5' deletion constructs retained the XbaI-KpnI-EcoRI multiple cloning site of pDdGal16. The 5' deletion constructs are designated y, where y refers to the nucleotide at the 5' deletion end point. Base +1 is the A residue in the initiation codon ATG.

To construct the internal deletions, two PCR-amplified fragments corresponding to the 5' upstream sequence of rnrB were cloned into the XbaI or XbaI/EcoRI sites of the 5' deletion constructs. The 5' primer for PCR, 5'-TTACTAGTGAAATACCTGCACCTCC-3', is complementary to the coding strand of capA, the gene adjacent to rnrB. It contains an added SpeI site for cloning into the XbaI site of the deletion plasmids. The two 3' primers, each bearing an EcoRI site, are oligonucleotides spanning either box B, 5'-TTGAATTCAAAATACACACACATTCCCG-3', or box C, 5'-TTGAATTCATGATGGAATCACCGTTCC-3' (Fig. 1B). Polymerase chain reaction was performed with Expand^TM (Roche Molecular Biochemicals) according to the manufacturer's instructions with the annealing temperature set at 55 °C. The internal deletions are designated xy, where x and y indicate the nucleotide at the end point of the 3' and 5' deletions, respectively.

For construct 444212, one of the PCR products was digested with SpeI and XbaI and inserted into the XbaI site of construct 212. All other internal deletion constructs do not contain sequences 5' from the XbaI site. Deletion 292212 was constructed by inserting the XbaI-EcoRI restriction fragment from the product generated with the PCR primer spanning box B into XbaI-EcoRI of construct 212, while deletion 359280 combined the construct 280 with the XbaI-EcoRI fragment from the PCR product generated with the primer spanning box C.

For the remaining constructs, individual GC-rich boxes were made with pairs of oligonucleotides designed in such a way that, after annealing, there would be on both sides overhanging ends, GATC, that are compatible with a BglII site. The sequences of the oligonucleotides for reconstituting the boxes are as follows: for box B, 5'-GATCCTTTCGGGAATGTGTGTGTATTA-3' and 5'-GATCTAATACACACACATTCCCGAAAG-3'; for box C, 5'-GATCCATTGGAACGGTGATTCCATCAA-3' and 5'-GATCTTGATGGAATCACCGTTCCAATG-3'; and for box D, 5'-GATCCTCTAGAATCGGAGTGGTACCCAAAA-3' and 5'-GATCTTTTGGGTACCACTCCGATTCTAGAG-3'. The recipient plasmids were 340, 280, and 212. To accommodate the GATC overhang of the annealed oligonucleotides, the XbaI-KpnI-EcoRI sites in the recipient plasmids were replaced with SpeI-BglII-EcoRI, obtained from the multiple cloning site of the vector pPC86 (32). Constructs 430340 and D212 were made by combining box D with the modified 340 and 212, respectively. Constructs B212 and C212 were generated by placing boxes B and C in the modified 212.

Dot-Blot Analysis-- The accumulation of transcript was quantified using dot-blot analysis because on RNA gel the lacZ fusion transcript migrated as a smear similar to that observed in other studies (33, 34). Total cellular RNA was extracted according to Franke et al. (35), treated with RQ1 RNase-free DNase (Promega), and quantified by spectrophotometry. Two dots containing 5 µg of RNA each were spotted onto Nytran membrane (Schleicher & Schuell). Expression of the rnrB-lacZ fusion was determined by probing the membranes with the lacZ gene using the HindIII-XhoI fragment from pDdGal16. For endogenous rnrB expression, the EcoRI-DraI fragment corresponding to the region of rnrB that was not present in the fusion constructs (18) was used as a probe. All probes were labeled by random priming (31). For the lacZ probe the membranes were hybridized in Denhardt's solution with 50% formamide for 16 h at 45 °C and washed twice for 20 min each in 0.1× SSC, 0.1% SDS at 65 °C. For the rnrB probe, hybridization was conducted at 40 °C for 16 h and washed twice for 20 min each in 1× SSC, 0.1% SDS at 65 °C. These conditions were determined by Northern blotting to retain specific signals with undetectable background (data not shown). The blots were exposed to a -imaging screen on a phosphor imager GS-363 (Bio-Rad), and the levels of expression were quantified with Molecular Analyst^TM software (Bio-Rad).

Histological Staining-- Cells were grown on bacteria and developed on preboiled nitrocellulose filters resting on KKP-saturated pads at a density of about 2 × 10⁶ cells/cm². At the slug stage, the filters supporting the slugs were fixed in 0.1% glutaraldehyde in Z buffer for 10 min and assayed for -galactosidase activity (36). The reaction was stopped with 3% trichloroacetic acid. The filters were washed in water, mounted on microscopic slides under coverslips, and examined on a Zeiss Axiophot microscope with a × 10 objective. Pictures were taken on Kodak Royal Gold ASA 25 film.

Electrophoretic Mobility Shift Assays-- The cytosolic and nuclear extracts were prepared as described (24). The probes were the oligonucleotides reconstituting boxes A and B as well as CAE-1 (21). The sequences for the box A duplex are 5'-GATCCATAGGAACCAAAATTGCGCTAA-3' and 5'-GATCTTAGCGCAATTTTGGTTCCTATG-3'. The sequences for the box B duplex are listed above. Unlabeled oligonucleotides corresponding to these sequences along with box C and box D duplexes, also shown above, were used in competition assays.

For the binding assays, 10 µg of protein extract were incubated with 3000 cpm of end-labeled probe (0.1-0.5 ng), 500 ng of double-stranded poly(dI-dC) (Sigma), and 1 µg of bovine serum albumin in a final volume of 20 µl of 0.25× storage buffer (24). The components were allowed to bind at room temperature for 30 min, immediately applied on a 4.5% acrylamide-TBE gel, and resolved at 4 °C at 140 V until the unbound probe reached the bottom of the gel. Following electrophoresis, the gel was fixed in 10% acetic acid, dried, and exposed to x-ray films (Kodak X-Omat).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Upstream Regions Regulating the Expression of rnrB-- Primer extension of the rnrB transcript showed a single product corresponding to 114 base pairs upstream of the ATG initiation codon. This site is preceded by a stretch of T residues that is typical of transcription start sites of D. discoideum genes (Fig. 1B) (37).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   5' upstream region of rnrB. A, schematic representation of the intergenic region between rnrB and capA. The checkered boxes show the protein coding regions including introns. The arrows indicate the direction of transcription. The G/C-rich sequences in the intergenic region are represented by stippled boxes. Also marked are locations of key restriction enzyme sites. B, sequence of the region upstream of the start codon of rnrB. The boxed sequences show the G/C-rich regions referred to as boxes A, B, C, and D. Borders of deletion constructs are marked by vertical lines. The vertical arrow indicates the position of the transcriptional start site.

Cells transformed with the 5' deletion construct 450 expressed lacZ mRNA with a developmental profile that was indistinguishable from the endogenous rnrB mRNA: moderate level in vegetative cells, low level in early developing cells, and a resurgence of activity in postaggregative cells (Figs. 2A and 3A). Histochemical staining also showed that the spatial expression of -galactosidase directed by this deletion construct is identical to that of the undeleted control. Therefore, subsequent deletion analysis focused on the region downstream of 450. As observed in other D. discoideum promoters, the sequence upstream of the rnrB coding region is made up primarily of A/T residues with G/C-rich clusters. The four G/C-rich clusters detected between the translation start codon and 450 are referred to as boxes A, B, C, and D (Fig. 1).

View larger version (64K): [in this window] [in a new window]   Fig. 2.   Dot blot analysis of the transcript levels of rnrB and lacZ. Total cellular RNA was extracted from vegetative cells (0 h) or cells developed for 5 or 16 h. The RNA was spotted in duplicate and analyzed by hybridization with either rnrB or lacZ probes. A, analysis of RNA prepared from cells transformed with 5' deletion constructs. B, analysis of RNA extracted from cells transformed with internal deletion constructs.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Summary of the resurgence of transcriptional activity during postaggregative development directed by the deletion constructs. Results of dot blots were quantified. We normalized the transcript level in postaggregative cells by dividing the transcript level in 15 h developing cells by the transcript level in vegetative cells. We scored as strong resurgence of transcriptional activity during postaggregative development (+) in cases where the normalized lacZ transcript level was at least 50% as high as the normalized rnrB transcript level. In cases where the lacZ level was between 25 and 50% that of the rnrB message in post-aggregative cells, the resurgence activity was defined as weak (+/). Finally, constructs directing lacZ levels that were less than 25% that of rnrB were recorded as having poor resurgence activity ().

Analysis of 5' Deletion Constructs-- Cells carrying constructs extending to 311 or further upstream accumulated lacZ RNA with a profile that was identical to the accumulation of endogenous rnrB mRNA (Figs. 2A and 3A). These results suggest that the sequence downstream of 311 is sufficient for both vegetative expression and late resurgence of the transcript. Removing the upstream sequence to 280 did not affect the expression of rnrB/lacZ in vegetative cells or in early developing cells, but the level of rnrB/lacZ transcript in postaggregative cells was about half that of deletion 311. Truncation of the 5' sequence to 212 eliminated all activity in postaggregative cells, but the regulation in vegetative and early developing cells appeared unaffected (Figs. 2A and 3A). These results suggest that the sequences encompassing box A, between 280 and 212, and box B, between 311 and 280, are involved in regulating the resurgent activity during late development.

While deletion 212 retained rnrB activity in vegetative cells, truncation to 130 abolished all activity (Figs. 2A and 3A), suggesting that the A/T-rich sequence between 130 and 212 is required for activity in vegetative cells.

Analysis of Internal Deletion Constructs-- Cells carrying construct 444212 did not exhibit activity in postaggregative cells (Figs. 2B and 3B). Together with results obtained from 5' deletion analysis, this suggests that the elements regulating the expression of rnrB during late development are located between 444 and 212. Subsequent analysis of internal deletions was focused on the region downstream of 444. Cells transformed with constructs missing box A (deletion 292212), box B (deletion 359280), or box C (construct 429340) exhibited a normal level of reporter transcript during late development (Figs. 2B and 3B). Cells transformed with construct 359212, missing boxes A and B, also expressed lacZ RNA during postaggregative development (Figs. 2B and 3B). Therefore, while the 5' deletion analysis shows that the region downstream of 311 is sufficient to promote a normal level of expression, results from internal deletions suggest that the region between 359 and 444 also has a positive effect on transcriptional activity during late development.

Elements Promoting Transcription during Late Development-- The 5' deletion analysis suggested that box A alone was able to drive expression, albeit at low levels, of lacZ RNA during late development. To assess the role of the other discrete elements, we fused oligonucleotides corresponding to the G/C-rich boxes B, C, and D to the minimal promoter 212. Cells transformed with construct B212, containing box B, showed a resurgence of activity. With construct C212, bearing box C, no activity was detected in developing cells. Cells carrying construct D212, containing box D, displayed a low level of activity in late developing cells (Figs. 2B and 3B). These results provide further evidence that sequences corresponding to boxes A, B, and D can independently activate transcription in late developing cells.

Spatial Localization of -Galactosidase Activity-- Previously, we showed by histochemical staining that cells bearing a plasmid containing the rnrB promoter fused to lacZ-expressed -galactosidase only in the posterior, prespore zone (18). Fig. 4 shows that deletion constructs that did not express lacZ RNA in postaggregative cells exhibited little or no -galactosidase activity in slugs (e.g. constructs 212, 444212, and C212 (Fig. 4, E, F, and L)). This indicates that the -galactosidase activity observed in the slugs is directed by lacZ transcript present during this stage of development and is not caused by residual activity held over from vegetative cells.

View larger version (55K): [in this window] [in a new window]   Fig. 4.   Histochemical staining of -galactosidase activity. Cells transformed with various rnrB/lacZ constructs were developed for 16-18 h to the migrating slug stage. The slugs were fixed and assayed for -galactosidase activity histochemically. A shows the staining pattern of a slug developed from cells that had been transformed with an undeleted construct. B-M display the staining patterns of slugs whose cells harbor the deletion constructs indicated. In each panel, the anterior or prestalk region of the slug is pointing toward the right. Also shown in the bottom part of each panel is the schematic presentation of the borders of the deletion.

Deletion constructs containing both box A and box B (311 and 429340) displayed prespore-specific expression of -galactosidase activity at the slug stage similar to that of the undeleted construct (Fig. 4, A, B, and I). Constructs carrying either box A or box B alone (280, 292212, 359280, and B212) exhibited -galactosidase activity primarily in the prespore zone with some activity in the prestalk region (Fig. 4, C, G, H, and K). However, slugs developed from cells carrying -225, containing the proximal half of box A, exhibited random distribution of -galactosidase activity (Fig. 4D).

The internal deletion constructs that are missing both boxes A and B, 359212 and D212, exhibited unexpected results. They directed the expression of lacZ RNA in postaggregative cells (Figs. 2B and 3B) but displayed a very low level of -galactosidase scattered throughout the slugs (Fig. 4, J and M). These results suggest that the RNA expressed by these two constructs during late development is randomly distributed in the slug and is not efficiently translated into -galactosidase.

Factors Interacting with the cis-Regulatory Elements-- Results from the deletion analysis revealed that box A and box B are important for spatial and temporal control of rnrB expression during late development. We performed electrophoretic mobility shift assays using oligonucleotides containing these sequences to identify cellular factors that recognize these elements.

Fig. 5A shows the specificity of binding of an oligonucleotide containing box A to factors in nuclear extracts. A 300-fold molar excess of oligonucleotides corresponding to either box B, box C, box D, or CAE-1 did not compete for binding of box A in the fast migrating complex. The factor that interacted with box A specifically was undetectable in nuclear extracts prepared from vegetative cells or from cells developed for 6 h. It was present in cells developed for 12, 15, and 18 h (Fig. 5B). The presence of this activity in cytosolic extracts could not be assessed because of the presence of a nonspecific complex with similar mobility on the gel shift assay (data not shown).

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Electrophoretic mobility shift assay of nuclear extracts for factors binding to the box A oligonucleotide. A, mobility shift of the box A oligonucleotide probe in the presence of unlabeled oligonucleotides. Nuclear extract prepared from cells that had been developed for 15 h was mixed with labeled oligonucleotide corresponding to box A (far left lane) or mixed with labeled box A oligonucleotide in the presence of excess unlabeled oligonucleotides corresponding to box A, box B, box C, box D, or the CAE-1 element. The far right lane shows the migration of the labeled box A probe in the absence of nuclear extract. B, developmental profile of the binding factor. Nuclear extracts were prepared from vegetative cells, 0 h, or from cells that had been developed for 6, 12, 15, or 18 h. The nuclear extracts were mixed with labeled oligonucleotide corresponding to box A and resolved on a polyacrylamide gel. The arrow marks the position of the specific complex.

Fig. 6A shows the presence of a cytosolic factor that exhibits specific binding to the box B oligonucleotide. This factor was effectively competed with unlabeled box B but poorly competed with unlabeled box A, C, or D. It was present in cytosolic extracts from vegetatively growing cells as well as cells from all stages of development (Fig. 6B). A complex of box B with the same electrophoretic mobility was detected in nuclear extracts. This factor was not detected in the nuclear fractions of vegetative cells or early developing cells, but it was present in the nuclear extracts of cells that had been developed for 12 h or more (Fig. 6C).

View larger version (52K): [in this window] [in a new window]   Fig. 6.   Electrophoretic mobility shift assay for factors binding to the box B oligonucleotide. A, mobility shift of the box B oligonucleotide probe in the presence of unlabeled oligonucleotides. Cytosolic fraction prepared from cells that had been developed for 18 h was mixed with labeled oligonucleotide corresponding to box B (far left lane) or mixed with labeled box B oligonucleotide in the presence of excess unlabeled oligonucleotides corresponding to box B, CAE-1, box A, or box C. B, developmental profile of the binding factor. Cytosolic fractions were prepared from vegetative cells, 0 h, or from cells that had been developed for 6, 12, 15, or 18 h. The cytosolic fractions were mixed with labeled oligonucleotide corresponding to box B and resolved on a polyacrylamide gel. C, developmental profile of the binding factor in nuclear extracts. Nuclear extracts were prepared from vegetative cells, 0 h, or from cells that had been developed for 6, 12, 15, or 18 h. The nuclear extracts were mixed with labeled oligonucleotide corresponding to box B and resolved on a polyacrylamide gel. In all three panels the far right lane shows the migration of the labeled box B probe in the absence of cellular proteins. The arrow marks the position of the specific complex.

The sequence of box B harbors half of a CAE box found in promoters of several cAMP-induced genes (19, 22, 23, 33, 38-40). The CAEs interact specifically with the trans-acting factor GBF (41), with the element CAE-1 of cotC displaying the highest affinity (39). Competition experiments showed that a 300-fold excess of CAE-1 was about as effective a competitor as 30-fold box B (Fig. 6A, compare lanes 3 and 8). Moreover, box B was a poor competitor for the CAE-1 element (data not shown). These results suggest that the affinity of GBF for box B is low and that box B interacts specifically with a factor that is distinct from GBF.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The gene encoding the small subunit of ribonucleotide reductase, rnrB, of D. discoideum has an unusual pattern of expression. It is the only gene identified so far that is expressed during vegetative growth, down-regulated during early development, and then expressed at high level in a cell type-specific manner during postaggregative development. We show here that postaggregative expression of rnrB requires controlling elements that are not needed for expression in growing cells. We also did not find repressor elements that could cause a decrease in transcriptional activity during early development. These results therefore suggest that the mechanism regulating the expression of rnrB during growth is different from that controlling its expression during postaggregative development. They also imply that the low level of rnrB transcript present in early developing cells is the result of a reduced level of transcription and the degradation of transcript synthesized during growth.

cis-Regulatory Element Responsible for rnrB Expression in Vegetative Cells-- Deletion analysis of the rnrB promoter suggests that the region located between 212 and 130, with an A/T content of 96%, is required for expression in vegetative cells. Elements rich in A/T have also been implicated in the regulation of other D. discoideum genes expressed during vegetative growth (40, 42-46). Moreover, the A/T-rich upstream activating sequence of actin 15 when placed in a developmentally regulated promoter can direct expression in vegetative cells (42). Hence, the involvement of A/T-rich elements in regulation appears to be a common feature among D. discoideum genes expressed during vegetative growth. However, there is no apparent sequence similarity between the various A/T-rich elements identified, perhaps reflecting the diverse mechanisms of gene regulation during growth phase. Alternatively, these A/T-rich elements may fold into similar structures that interact with related factors.

cis-Regulatory Elements Responsible for Prespore Expression of rnrB-- How the expression of prespore-specific genes is regulated in D. discoideum is not well understood. Consensus CAEs are found in promoters of most prespore genes studied to date (19, 20, 47). In most instances, the consensus CAEs have been shown to influence the level of activity but do not confer cell type specificity. For example, removal of the CAEs from the promoter of prespore genes drastically reduces the level of expression but does not alter the spatial distribution of the residual activity (19-21, 47). Also consistent with this idea is the observed function of GBF, the transcriptional activator that interacts with CAEs. Binding sites of GBF are found in the promoters of both prespore and prestalk genes (19-21, 38, 47-49). Cells carrying a null mutation in GBF are arrested at the loose aggregate stage before cell type specification takes place (24). However, in certain contexts the CAEs have been shown to affect spatial expression. For example, mutations in one of three CAEs in the promoter of the prespore cotC gene can result in asymmetrical expression within the prespore zone (21, 22). Another case is that of the car3 gene, which is expressed throughout the slug, and its promoter harbors two CAEs. Deletion of either of the two CAEs restricts expression to the prespore zone (50). Taken together, these results suggest that interactions between GBF and transcriptional factors that bind cis-regulatory elements other than CAEs are involved in regulating the expression of cell type-specific genes. The other cis-regulatory elements that may be involved include the A/T-rich elements identified in promoters of several genes expressed in postaggregative cells (19, 20, 38, 43, 47, 51).

Results presented here suggest that the expression of rnrB in prespore cells involves mechanisms that are distinct from those regulating prespore-specific genes described previously. The rnrB promoter does not possess consensus CAEs. The three G/C-rich regions (boxes A, B, and D) implicated in the expression of rnrB in postaggregative cells bear no sequence similarity among themselves. Box D supports very low level of activity that is randomly distributed in the slug (Fig. 4M). Boxes A and B can independently influence expression in prespore cells during postaggregative development. However, constructs containing only one of the two boxes exhibit activity that is lower than those carrying both boxes. In addition, these constructs show ectopic expression with activity transgressing to the prestalk zone (Fig. 4, C, G, H, and K). Internal deletions missing both box A and box B display very low level of activity in postaggregative cells, and the residual activity is randomly distributed in the slug (Fig. 4, J and M). Moreover, deletion of part of box A results in expression throughout the entire slug (Fig. 4D). These results suggest that prespore-specific expression of rnrB depends on the presence of both box A and box B (Fig. 4, A, B, and I). Unlike the CAEs, therefore, intact box A and box B play a direct role in specifying prespore expression. We are presently testing whether the presence of A/T-rich element(s) is required for expression of the rnrB gene during development like in other postaggregative genes (19, 20, 38, 43, 47, 51).

Identification of trans-Acting Factors Involved in rnrB Expression-- Two transcriptional activators, GBF (24) and STAT (52), that regulate developmental genes of D. discoideum have been characterized. GBF binds with CAEs, while STAT has been shown to interact with an A/T-rich element of the prestalk gene ecmB. Electrophoretic mobility shift assays showed that box A binds to a factor that is present in the nuclear fraction. This factor appears between 6 and 12 h of development (Fig. 5B), a profile consistent with the timing of the resurgence of rnrB transcript (18). The binding of box A to this factor was not affected by the addition of excess CAE-1 (Fig. 5A) or the STAT-binding site (data not shown). These data suggest that box A interacts with a nuclear factor that has not yet been characterized.

Box B interacts with a factor that can be detected in the cytosolic fraction during growth and development. This factor is detected in the nuclear fraction of developing cells but not vegetative cells (Fig. 6B), suggesting that the factor migrates to the nucleus or is modified in the nucleus during development. Box B contains G/T repeats that resemble half of the consensus GBF-binding site. Results from competition assays suggest that box B interacts weakly with GBF (Fig. 6A). This observation implies that GBF does not have a major role in the regulation of rnrB expression.

	ACKNOWLEDGEMENTS

We thank Zeina Saikali and Abraham Shtevi for technical assistance.

	FOOTNOTES

^* 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.

^§ Present address: MethylGene Inc., 7220 Frederick-Banting, Ville St-Laurent, Quebec H4S 2A1, Canada.

^** Recipient of an NSERC postgraduate scholarship.

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

The abbreviations used are: CAE, C/A-rich element; PIPES, 1,4-piperazinediethanesulfonic acid; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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