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Volume 270, Number 26, Issue of June 30, pp. 15808-15814, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Nucleotide Sequence Essential for the Function of DRE, a Common Promoter Element for Drosophila DNA Replication-related Genes (*)

Masamitsu Yamaguchi (§) , Yuko Hayashi , Yoshio Nishimoto , Fumiko Hirose , Akio Matsukage

From the (1)Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, 464, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase 180-kDa catalytic subunit gene contain a common 8 base pair (bp) promoter element, 5`-TATCGATA (DRE, Drosophila DNA replication-related element). We have generated various base substitutions and internal deletions in and around DRE (nucleotide positions -93 to -100 with respect to the transcription initiation site) of the PCNA gene in vitro and subsequently examined their effects on the binding to DREF (DRE-binding factor) and PCNA gene promoter activity in cultured Drosophila Kc cells as well as in living flies. Gel mobility shift assays using nuclear extracts of Kc cells with and without competitor DNA fragments carrying the mutations indicated that the 10-bp sequence from positions -91 to -100 is essential for complex formation with DREF. Transient expression assays of chloramphenicol acetyltransferase (CAT) in Kc cells transfected with PCNA promoter-CAT fusion genes carrying the mutations revealed that the 8-bp sequence from -93 to -100 is essential for activation of the promoter in Kc cells. Examination of lacZ expression from PCNA promoter-lacZ fusion genes carrying the mutations, introduced into flies by germ-line transformation, revealed that the 8-bp sequence is also important for DRE function during development. However, we obtained two exceptional mutations in the 8-bp sequence that did not or only marginally affected the PCNA gene promoter activity in transgenic flies. Both of these mutations effectively reduced the promoter activity in CAT transient expression assay in Kc cells and the binding to DREF in vitro. Therefore, the 8-bp sequence requirement for DRE function appears to be less stringent in living flies than in the cultured cell or in vitro cases.

INTRODUCTION

DNA replication is one of the most important processes for cell proliferation. Many lines of evidence have indicated that the expression of genes involved in DNA replication is closely correlated with the proliferating state of cells and repressed in accordance with the progression of differentiation in various tissues during development(1, 2) .

In budding yeast, genes involved in DNA replication contain a common promoter element (MluI cell cycle box, 5`-ACGCGT) (3) and the specific transcription factor complex DSC1 (MBF) is required for expression at the G1-S boundary(4, 5) . In mammalian cells, expression of genes involved in DNA replication increase dramatically at late G1 in response to growth stimulation, although they only marginally increase at the G1-S boundary in cycling cells(6, 7) . Mammalian genes such as those encoding DNA polymerase , thymidine kinase, dihydrofolate reductase and proliferating cell nuclear antigen (PCNA)()contain the transcription factor E2F-binding site (5`-TTTCGCGC) within their essential promoter sequences(8, 9) , although a critical role for the site in regulated expression in late G1 has been noted only for the Chinese hamster dihydrofolate reductase gene(10) .

We have isolated Drosophila genes for PCNA (11) and the DNA polymerase 180-kDa catalytic subunit (12) and found unique sequences (DNA replication-related elements (DRE)) containing a common 8-base pair (bp) palindromic sequence (5`-TATCGATA). Three DREs and one DRE are present in the DNA polymerase gene (at nucleotide positions -217, -83, and -30 with respect to the transcription initiation site) and in the PCNA gene(-100), respectively. Transient expression assays in cultured Kc cells have indicated that DRE functions as a positive cis-acting element(13) . DRE stimulated the activity of the heterologous promoter of the Drosophila metallothionein gene, in addition to the promoter of the PCNA gene, when it was placed upstream from these promoters in a normal or a reverse orientation (13). We also found a specific DRE-binding protein factor, DREF, consisting of a 86-kDa polypeptide homodimer(13) . DNase I footprinting analysis indicated that DREF binds to the 24-bp DRE region in which the 8-bp palindromic sequence is centered(13) . Although the 8-bp palindromic sequence is conserved between DRE regions of PCNA and DNA polymerase genes, the precise nucleotide sequence essential for DRE function has not been determined. Such a determination is important to identify DREs in promoter regions of other Drosophila genes and in those of DNA replication-related genes of other organisms. This would be an initial step to generalize our finding of the DRE-DREF system(14) . Furthermore, a role for DRE in the regulation of expression of genes involved in DNA replication has not yet been confirmed in living flies, and transgenic Drosophila provides an appropriate system to characterize transcriptional regulatory elements in vivo.

In the present study, we therefore generated various mutations in and around the DRE of the PCNA gene in vitro and subsequently examined their effects on binding to DREF and DRE function in cultured Kc cells as well as in living flies. We found that a 10-bp sequence including the 8-bp palindromic sequence is necessary for binding to DREF. In Kc cells, the 8-bp palindromic sequence is necessary and sufficient for the DRE function. In living flies, the 8-bp sequence is important for the DRE function, but its requirement appears to be less stringent than that in cultured cells.

EXPERIMENTAL PROCEDURES

Oligonucleotides

The chemically synthesized oligonucleotides used are as follows: SalI(-168), 5`-GGTTGTCGACTTTGAAATAAATATACTCTGT; AR, 5`-TCTATCGATAGCAGTAAGCGAGCGGCCTGC; BR, 5`-TCTATCGATAGCCTGCAGCGAGCGGCCTGC; CR, 5`-TCTATCGATATAAGGCAGCGAGCGGCCT; DR, 5`-TCTATCGAGCGCAGGCAGCGAGCGGCCT; EN, 5`-CGAGCGATTCAGGCGAT; ER, 5`-ATCGCCTGAATCGCT; FN, 5`-CGATATCTTCAGGCGAT; FR, 5`-ATCGCCTGAAGATAT; GN, 5`-CGATAGAGGCAGGCGAT; GR, 5`-ATCGCCTGCCTCTAT; HN, 5`-CGATAGATTACGGCGAT; HR, 5`-ATCGCCGTAATCTAT.

The double-stranded 30-bp oligonucleotide, DRE-P contains the 24-bp DRE sequence of the PCNA gene promoter and the 6-bp linker sequence(13) .

Plasmid Constructions

The plasmid p5`-168DPCNACAT contains the PCNA gene fragment spanning from -168 to +23 placed upstream of the chloramphenicol acetyltransferase (CAT) gene in the plasmid pSKCAT (15). p5`-168ClaI(-)DPCNACAT contains a 2-bp GC insertion at position -96 of the PCNA gene promoter(13) .

A fragment from -91 to -168 having a 2-bp substitutional mutation was generated by the polymerase chain reaction (PCR) method (16) using p5`-168DPCNACAT as a template with primers SalI(-168) and AR. The PCR product was digested with SalI and ClaI, then replaced with the fragment between SalI and ClaI sites of the p5`-168DPCNACAT to create the plasmid p5`-168 mutADPCNACAT. Plasmids p5`-168 mutBDPCNACAT, p5`-168 mutCDPCNACAT, and p5`-168 mutDDPCNACAT were constructed in the same way except that BR, CR, and DR in addition to SalI(-168) were used as PCR primers, respectively. In constructing p5`-168 mutDDPCNACAT, the PCR product was digested with SalI and TaqI instead of SalI and ClaI.

A double-stranded oligonucleotide was obtained by annealing the oligonucleotides EN and ER, then placing the fragment between ClaI and EcoRV sites of the p5`-168DPCNACAT to create the plasmid p5`-168 mutEDPCNACAT. Plasmids p5`-168mutFDPCNACAT, p5`-168 mutGDPCNACAT, and p5`-168 mutHDPCNACAT were constructed in the same way using oligonucleotides FN/FR, GN/GR, and HN/HR, respectively. The obtained plasmids were verified by nucleotide sequence analysis with synthetic primers(17) .

To prepare internal deletions, p5`-168DPCNACAT was digested with ClaI, then with mung-bean nuclease, followed by self-ligation using T4 DNA ligase. Various internal deletions were obtained, although the procedures have been considered to produce a 2-bp deletion at the ClaI site. The obtained deletion derivatives determined by nucleotide sequence analysis are as follows; p5`-168 mut1(-95)DPCNACAT having a 1-bp deletion at position -95, p5`-168mut1(-96)DPCNACAT having a 1-bp deletion at position -96, p5`-168mut1(-98)DPCNACAT having a 1-bp deletion at position -98, p5`-168mut3DPCNACAT having a 3-bp deletion from -95 to -97, p5`-168mut4DPCNACAT having a 4-bp deletion from -95 to -98, p5`-168mut6DPCNACAT having a 6-bp deletion from -94 to -99, p5`-168mut18DPCNACAT having a 18-bp deletion from -81 to -98.

The plasmid p5`-607DPCNAlacZW8HS (15) contains the PCNA gene fragment spanning from -607 to +137 fused with the lacZ in a P-element vector. The plasmid p5`-168DPCNAlacZW8HS (15) contains the PCNA gene fragment spanning from -168 to +137 fused with the lacZ in a P-element vector. To create mutated derivatives in P-element vector backbones, fragments having various mutations in and around DRE were isolated from CAT plasmids by digestion with SalI(-168) and SacII (+23), then inserted between XhoI(-607) and SacII (+23) sites of the p5`-607DPCNAlacZW8HS. The obtained plasmids were verified by nucleotide sequence analysis with synthetic primers.

Drosophila hsp70 gene promoter (18) was isolated from the pDhsp70GEM3 by digestion with KpnI and HindIII, and then inserted between KpnI and HindIII sites of pGV-B (Toyo Ink) carrying the luciferase gene to create pDhsp70-L.

All plasmids were propagated in Escherichia coli XL-1 Blue and isolated by standard procedures(19) . The isolated plasmids were further purified through two cycles of ethidium bromide/CsCl density-gradient centrifugation.

Preparation of Nuclear Extracts and Gel Mobility Shift Assay

Preparation of nuclear extracts from Kc cells was as described elsewhere(13) . Each nuclear extract was incubated in 15 µl of the reaction mixture containing 15 mM HEPES, pH 7.6, 60 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 12% glycerol, 1 µg of poly(dIdC) for 10 min on ice. Unlabeled competitor DNA fragments were added at this step. Then, P-end-labeled DRE-P oligonucleotides (40 pg, 1 10 counts/min) were added, and the mixture was incubated for 10 min on ice. The complex of DNA and DREF was electrophoretically separated from free probes in a 4% polyacrylamide gel in 50 mM Tris borate, pH 8.3, 1 mM EDTA containing 2.5% glycerol at room temperature. The gel was dried and autoradiographed. Competitor DNA fragments were isolated from PCNA gene promoter-CAT plasmids by digestion with SalI(-168) and EcoRV(-80).

DNA Transfection into Cells, CAT Assay, and Luciferase Assay

Drosophila Kc cells (20) were grown in M3(BF) medium supplemented with 2% fetal calf serum(21) . Cells were plated at about 5 10 cells/60-mm dish at 16 h before DNA transfection. DNA was transfected into cells by the calcium phosphate coprecipitation technique described elsewhere(22) . Each transfection contained 1 µg of PCNA gene promoter-CAT plasmid, 0.1 µg of pDhsp70-L, and 9 µg of pGEM3/dish. Cells were harvested at 48 h after transfection. Cell extracts were prepared, and CAT activity was measured as described previously(23) . The radioactivity of acetylated chloramphenicol on thin layer plates was quantified with an imaging analyzer BAS2000 (Fuji Film). The luciferase assay was carried out by means of a PicaGene assay kit (Toyo Ink) as described previously (9). All assays were performed within the range of linear relation of the activities to incubation time and protein amounts. CAT activity was normalized to the luciferase activity. The obtained values were essentially similar to those normalized to protein amounts which were determined by Bio-Rad protein assay. Transfections were performed several times with at least two independent plasmid preparations.

Establishment of Transgenic Flies

Fly stocks were maintained at 25 °C on standard food. Canton S flies were used as a wild-type strain. P-element-mediated germ line transformation was carried out as described(24) . G1 transformants were selected on the basis of white eye color rescue(25) . Multiple independent lines were obtained for each of the various fusion genes. Established transgenic fly strains and their chromosomal linkages are listed in . Occasionally, we obtained lines whose lacZ expression patterns were totally different from those of other lines carrying the same fusion genes (marked in ). Since they were very likely to be enhancer (promoter)-trapped lines, we did not count them in the present study.

Analysis of Expression Patterns for PCNA-lacZ

Quantitative measurement of -galactosidase activity in extracts was carried out as described(26) . Male transgenic flies were crossed with female wild-type flies. Groups of 50-100 individuals each of dechorionated embryos, larvae, pupae, and adult flies were homogenized in 500 µl of ice-cold assay buffer (50 mM potassium phosphate, pH 7.5, 1 mM MgCl). Homogenates were centrifuged at 10,000 g at 4 °C for 5 min. For each assay, 50-200 µl of the supernatant was added to give 1 ml of assay buffer containing 1 mM chlorophenol red--D-galactopyranoside substrate (CPRG; Boehringer Mannheim). Reaction incubations were at 37 °C in the dark. Substrate conversion was measured at 574 nm using a spectrophotometer at 0.25, 0.5, 0.75, 1, and 1.5 h after addition of the extract, and the rate of color development was linear. The -galactosidase activity was defined as absorbance units/hour/milligram of protein. To correct for endogenous -galactosidase activity, extracts from the wild-type strain were included in each experiment, and this background reading was subtracted from readings obtained with each transformant line. Deviation among independent strains was less than 30% (not shown). The protein concentration of the extract was determined by Bio-Rad protein assay.

RESULTS

Effects of Mutations in and around the DRE Sequence on Binding to DREF

A set of base substitutions and deletions in and around the DRE sequence of the PCNA gene promoter was generated by in vitro mutagenesis (Fig. 1B). Subsequently, we examined the effects of these mutations on the binding to DREF. Nuclear extracts were prepared from Kc cells, and gel mobility shift assays were carried out. As noted previously(13) , a specific DNAprotein complex could be detected using a chemically synthesized DRE-P oligonucleotide as a probe (Fig. 2, A-C, lanes b and m). The band shifted with P-labeled DRE-P was diminished by adding an excess amount of unlabeled DRE-P as a competitor, but not by adding an unrelated sequence of similar size(13) . Furthermore, one of the monoclonal antibodies against DREF (mAb#1) diminished the shifted band and the other (mAb#4) super-shifted the band (data not shown). Therefore, the shifted band appears to represent the DREDREF complex.

Figure 1: Base substitution and internal deletion mutants in and around the DRE of the PCNA gene. A, constructs of wild-type PCNA-lacZ and PCNA-CAT fusion genes. The vertical line with a horizontal arrow indicates the transcription initiation site. The open and closed boxes indicate the 5`-untranslated and coding sequences of the PCNA gene, respectively. The dark stippled boxes indicate the DRE sequence. The shaded and the hatched boxes indicate the lacZ coding and CAT coding sequences, respectively. B, nucleotide sequences in and around DRE of wild-type and mutant PCNA genes. Nucleotide sequences with substitution for the wild-type sequence are shown by small letters and those deleted are shown by dotted lines. The inserted nucleotide sequence is also shown. The pair of arrows (head to head) indicates the palindromic sequence. The closed box indicates the 8-bp DRE sequence. The region required to compete for the formation of DREDREF complex in the gel mobility shift assay is indicated by the bracket above.


Figure 2: Competition for complex formation between the DRE-P oligonucleotide and Kc cell nuclear extract. Radiolabeled double-stranded DRE-P oligonucleotides were incubated with or without (-) nuclear extract (2 µg of protein) in the presence or absence (0) of indicated amounts of competitor DNA fragments (from -80 to -168; indicated at the top of each panel). Gel mobility shift assays shown in panels A-C were performed independently, and competition with the wild-type fragment was included for each experiment as a control.


DNA fragments (from -80 to -168) carrying various mutations (Fig. 1B) were added to the binding reaction as competitors. As shown in Fig. 2, A and B, fragments mutA, mutB, mutC, mutG, and mutH carrying base substitutions outside the 8-bp palindromic sequence competed for the binding as effectively as the wild-type fragment. In contrast, fragments mutD and mutE carrying base substitutions inside the 8-bp sequence did not compete under the examined concentrations (Fig. 2A, lanes t-v; Fig. 2C, lanes f-h). Similarly, no competition was observed with the fragment mutF, although it contains base substitutions outside but adjacent to the 8-bp sequence (Fig. 2B, lanes f-h). DNA fragments carrying various deletions or a 2-base insertion in the 8-bp sequence did not compete for the binding to DREF (Fig. 2, B and C). From these results, taken together, it is concluded that the 10-bp sequence from -91 to -100 is essential for the formation of the complex with DREF.

Effects of Mutations in and around the DRE Sequence on PCNA Gene Promoter Activity in Cultured Kc Cells

The PCNA gene promoter carrying various mutations in and around DRE was placed upstream of the CAT gene in a CAT vector. Plasmids carrying these constructs were transfected into Kc cells and CAT expression levels were determined. As shown in Fig. 3, plasmids carrying base substitutions outside the 8-bp palindromic sequence showed no (panel A, lanes g-l and panel B, lanes g and h) or moderate reduction (panel B, lanes c-f) of CAT expression as compared with that of the original plasmid, p5`-168DPCNACAT (-168wt). In contrast, much more extensive reduction of CAT expression was observed with plasmids p5`-168mutDDPCNACAT (mutD) and p5`-168mutEDPCNACAT (mutE), both carrying base-substitutions within the 8-bp sequence (Fig. 3A, lanes e and f; Fig. 3B, lanes i and j). Similarly, a 1-bp deletion or a 2-bp insertion in the 8-bp sequence led to an extensive reduction of CAT expression (Fig. 3C, lanes c-f, m, and n). Deletions of more than 3-bp in the 8-bp sequence almost completely diminished CAT expression (Fig. 3B, lanes k and l; Fig. 3C, lanes g-l). These results indicate that the 8-bp sequence from -93 to -100 is essential for the promoter activity of the PCNA gene in Kc cells, and the 4-bp sequence from -87 to -90 might play an additional role in the promoter activity.

Figure 3: Effects of mutations in and around DRE on PCNA gene promoter activity in Kc cells. CAT plasmids harboring wild-type or mutant PCNA promoters (indicated at the top of each panel) were cotransfected with pDhsp70-L plasmid into Kc cells. 48 h after the transfection, cell extracts were prepared to determine the CAT expression levels. CAT activity was normalized to the luciferase activity. Averaged values obtained from several independent experiments with standard deviations are given as CAT activity relative to that of p5`-168DPCNACAT (-168wt, lanes a and b). Panels A-C show independent experiments, and wild-type PCNA-CAT (-168wt) and promoterless CAT (pSKCAT) plasmids were included as controls. Exposure time of the autoradiogram shown in panel B is shorter than those shown in panels A and C. Acetylated and non-acetylated forms of [C]chloramphenicol are marked by Ac and CM, respectively.


Effects of Mutations in and around the DRE Sequence on PCNA Gene Promoter Activity in Living Flies

We have established transgenic flies carrying PCNA (-168 to +137) and lacZ fusion genes (15). Male transgenic flies were crossed with wild-type females to examine zygotic expression of the lacZ. Expression of lacZ is high in embryos, first and second instar larvae, and adult females, and low at other stages of development(27) . This pattern of expression is very similar to the levels of zygotically expressed PCNA mRNA during development(11) . Spatial patterns of lacZ expression in the embryo visualized by immunostaining with anti-lacZ antibody were also similar to the distribution of the endogenous PCNA protein(27) .

To determine the essential sequence for DRE function during Drosophila development, we generated PCNA-lacZ fusion genes carrying internal deletions or base substitutions in and around DRE. These fusion genes were then introduced into flies by germ-line transformation. Established transgenic fly strains and their chromosomal linkages are listed in . Activities of modified promoters were monitored by the quantitative -galactosidase assay at various developmental stages of Drosophila. Two-bp substitutions outside the 8-bp palindromic sequence caused no appreciable reduction of lacZ expression throughout development (Fig. 4, mutA-C and F-H). In contrast, base substitutions inside the 8-bp sequence resulted in extensive reduction of lacZ expression in embryos and larvae, although high expression of the lacZ was still observed in adult females (Fig. 4, mutD and E). Similarly, a 1-bp deletion at position -96 and deletions more than 3-bp in the 8-bp palindromic sequence resulted in extensive reduction of lacZ expression in embryos and larvae (Fig. 5, mut1(-96), 3, 6, and 18). Here, too, high expression of the lacZ in adult females was still observed. However, surprisingly, only a moderate reduction of lacZ expression was observed with the transgenic strain carrying a 1-bp deletion at position -98 and almost no reduction was observed with the strain carrying a 2-bp insertion in the 8-bp palindromic sequence (Fig. 5, mut1(-98) and ClaI(-)). These are not likely to be due to chromosomal position effects, since six independent strains for each construct showed similar expression patterns. Therefore, the 8-bp palindromic sequence appears to be essential for DRE function that is required for the PCNA gene promoter activity in both embryos and larvae, although mutations in the 8-bp sequence can be tolerated under some conditions.

Figure 4: Effects of 2-bp substitution mutations in and around DRE on PCNA gene promoter activity in transgenic flies. Male transgenic flies (indicated in each panel) were crossed with female wild-type flies, and extracts were prepared from Drosophila bodies at various stages of development. The -galactosidase activities in the extracts are expressed as absorbance units/hour/milligram protein. Closed bars indicate the average values for independent transgenic strains carrying the indicated fusion gene. Numbers (n) of independent strains carrying the same fusion gene are given in each panel. mutA, strains carrying the p5`-168 mutADPCNAlacZW8HS; mutB, strains carrying the p5`-168 mutBDPCNAlacZW8HS; mutC, strains carrying the p5`-168 mutCDPCNAlacZW8HS; mutD, strains carrying the p5`-168 mutDDPCNAlacZW8HS; mutE, strains carrying the p5`-168 mutEDPCNAlacZW8HS; mutF, strains carrying the p5`-168 mutFDPCNAlacZW8HS; mutG, strains carrying the p5`-168 mutGDPCNAlacZW8HS; mutH, strains carrying the p5`-168 mutHDPCNAlacZW8HS.


Figure 5: Effects of internal deletion or insertion mutations in and around DRE on PCNA gene promoter activity in transgenic flies. Male transgenic flies (indicated in each panel) were crossed with female wild-type flies, and extracts were prepared from Drosophila bodies at various stages of development. The -galactosidase activities in the extracts are expressed as absorbance units/hour/milligram protein. Closed bars indicate the average values for independent transgenic strains carrying the indicated fusion gene. Numbers (n) of independent strains carrying the same fusion gene are given in each panel. mut3, strains carrying the p5`-168mut3DPCNAlacZW8HS; mut6, strains carrying the p5`-168mut6DPCNAlacZW8HS; mut18, strains carrying the p5`-168mut18DPCNAlacZW8HS; mut1(-96), strains carrying the p5`-168 mut1(-96)DPCNAlacZW8HS; mut1(-98), strains carrying the p5`-168mut1(-98)DPCNAlacZW8HS; ClaI(-), strains carrying the p5`-168ClaI(-)DPCNAlacZW8HS.


DISCUSSION

In our previous studies on Drosophila genes for PCNA and DNA polymerase 180-kDa catalytic subunit, we found a common regulatory element, DRE and a specific DREF(13) . DNase I footprinting analysis using Kc cell nuclear extracts indicated that DREF binds to the 24-bp DRE region in which the 8-bp palindromic sequence is centered (13). Methylation interference analysis using a 30-bp DRE oligonucleotide as a probe also indicated that 17 nucleotide residues in each strand can interact with DREF (data not shown). In the present study, we performed a gel mobility shift assay using Kc cell nuclear extracts and a 30-bp DRE-P oligonucleotide as a probe. We added 89-bp DNA fragments (from -168 to -80) carrying various mutations in and around DRE in the binding reaction as competitors. From this analysis, the nucleotide sequence essential for the binding to DREF was determined to be a 10-bp sequence which is significantly shorter than that suggested by previous studies. Very recently, we have detected a protein factor(s) that binds to the upstream and adjacent region of DRE in the PCNA gene promoter (data not shown). Since competitor DNAs used in the gel mobility shift assay contain a binding site for this factor in addition to DRE, molecular interaction of this factor with DREF might change the nature of the interaction between DREF and the DRE sequence.

Previously, we suggested that both the 8-bp palindromic sequence (5`-TATCGATA) and 2 thymidylate residues located 2 bp away from the palindromic sequence are important for DRE function, since one of the three DREs in DNA polymerase gene promoter in which an AC sequence is present in place of TT contributes much less to promoter activation than the other two(13) . However, the present detailed analysis with CAT transient expression assay and with transgenic flies clearly indicates that the 8-bp palindromic sequence without any additional sequence is essential and sufficient for DRE function. Based on the results, it is clearly of interest to identify the 8-bp palindromic sequence in the promoter regions of other DNA replication-related genes. We have searched for the 8-bp sequence in a Drosophila DNA data base and found that 60 copies are present within 600 bp upstream regions of transcription initiation sites of 49 genes. Interestingly, most of these genes are related to cell proliferation (a more detailed description will be published elsewhere). Therefore, it seems likely that DRE is a common regulatory element responsible for the coordinated regulation of proliferation-related genes in Drosophila.

In the analysis with transgenic flies, we found two exceptional mutations in the 8-bp palindromic sequence that did not or only marginally affected the PCNA gene promoter activity. In contrast, both of these mutations effectively reduced the promoter activity in CAT transient expression assay in Kc cells and the binding to DREF in vitro. The discrepancy may be explained by several possibilities. For instance, naked linear DNA used as a probe and competitors in gel mobility shift assay might not be able to reproduce faithfully the interaction of a transcription factor(s) and chromosomal DNA in vivo. Furthermore, it is known that high copy numbers (>10) of plasmid DNA are incorporated into cells by the calcium phosphate coprecipitation method, and most of them exist in an episomal state in cells. In contrast, the P-element method provides only one copy of the transgene integrated into near random places of the chromosomes. Therefore, the difference in results between transient expression and transgenic fly analyses might reflect differences in the ratios of DRE to DREF in cells, that is, a moderate reduction of affinity to DREF by some mutations in the 8-bp sequence might be overcome by an excess amount of DREF present in cells of the transgenic fly. In any event, the requirement for the 8-bp palindromic sequence for PCNA gene promoter activity appears to be less stringent in living flies than in cultured cells or in vitro. Further analysis is necessary to clarify this point.

  
Table: Transformants carrying the lacZ fused to the PCNA gene 5`-flanking sequences


FOOTNOTES

*
This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 81-52-762-6111 (ext. 8956); Fax: 81-52-763-5233.

The abbreviations used are: PCNA, proliferating cell nuclear antigen; DRE, replication-related element; DREF, DRE-binding factor; bp, base pair(s); CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; wt, wild-type.

ACKNOWLEDGEMENTS

We thank Noriko Hayashi for technical assistance. We also thank Malcolm Moore for comments on the manuscript.

REFERENCES
  1. Matsukage, A., Kitani, H., Yamaguchi, M., Kusakabe, M., Morita, T., and Koshida, Y. (1986) Dev. Biol.117, 226-232 [CrossRef][Medline] [Order article via Infotrieve]
  2. Matsukage, A., Hirose, F., and Yamaguchi, M. (1994) Jpn. J. Cancer Res.85, 1-8 [CrossRef][Medline] [Order article via Infotrieve]
  3. Lowndes, N. F., Johnson, A. L., and Johnston, L. H. (1991) Nature350, 247-250 [CrossRef][Medline] [Order article via Infotrieve]
  4. Dirick, L., Moll, T., Auer, H., and Nasmyth, K. (1992) Nature357, 508-513 [CrossRef][Medline] [Order article via Infotrieve]
  5. Lowndes, N. F., Johnston, A. L., Breeden, L., and Johnston, L. H. (1992) Nature357, 505-508 [CrossRef][Medline] [Order article via Infotrieve]
  6. Baserga, R. (1991) J. Cell Sci.98, 433-436 [Free Full Text]
  7. Miyazawa, H., Izumi, M., Tada, S., Takada, R., Masutani, M., Ui, M., and Hanaoka, F. (1993) J. Biol. Chem.268, 8111-8122 [Abstract/Free Full Text]
  8. Nevins, J. R. (1992) Science258, 424-429 [Abstract/Free Full Text]
  9. Yamaguchi, M., Hayashi, Y., Matsuoka, S., Takahashi, T., and Matsukage, A. (1994) Eur. J. Biochem.221, 227-237 [Medline] [Order article via Infotrieve]
  10. Slansky, J. E., Li, Y., Kaelin, W. G., and Farnham, P. J. (1993) Mol. Cell. Biol.13, 1610-1618 [Abstract/Free Full Text]
  11. Yamaguchi, M., Nishida, Y., Moriuchi, T., Hirose, F., Hui, C.-C., Suzuki, Y., and Matsukage, A. (1990) Mol. Cell. Biol.10, 872-879 [Abstract/Free Full Text]
  12. Hirose, F., Yamaguchi, M., Nishida, Y., Masutani, M., Miyazawa, H., Hanaoka, F., and Matsukage, A. (1991) Nucleic Acids Res.19, 4991-4998 [Abstract/Free Full Text]
  13. Hirose, F., Yamaguchi, M., Handa, H., Inomata, Y., and Matsukage, A. (1993) J. Biol. Chem.268, 2092-2099 [Abstract/Free Full Text]
  14. Hirose, F., Yamaguchi, M., and Matsukage, A. (1994) J. Biol. Chem.269, 2937-2942 [Abstract/Free Full Text]
  15. Yamaguchi, M., Hirose, F., Nishida, Y., and Matsukage, A. (1991) Mol. Cell. Biol.11, 4909-4917 [Abstract/Free Full Text]
  16. Kawasaki, E. S. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) pp. 21-27, Academic Press, San Diego, CA
  17. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A.74, 5463-5467 [Abstract/Free Full Text]
  18. Karch, F., Torok, I., and Tissieres, A. (1981) J. Mol. Biol.148, 219-230 [CrossRef][Medline] [Order article via Infotrieve]
  19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  20. Echalier, G., and Ohanessian, A. (1970) In Vitro6, 162-172 [Medline] [Order article via Infotrieve]
  21. Cross, D. P., and Sang, J. H. (1978) J. Embryol. Exp. Morphol.45, 161-172 [Medline] [Order article via Infotrieve]
  22. Di Nocera, P. P., and Dawid, I. B. (1983) Proc. Natl. Acad. Sci. U. S. A.80, 7095-7098 [Abstract/Free Full Text]
  23. Yamaguchi, M., Hayashi, Y., and Matsukage, A. (1988) Nucleic Acids Res.16, 8773-8787 [Abstract/Free Full Text]
  24. Spradling, A. C. (1986) in Drosophila: A Practical Approach (Roberts, D. B., ed) pp. 175-197, IRL Press, Oxford
  25. Robertson, H. M., Preston C. R., Philips, R. W., Johnson-Schlitz, D. M., Benz, W. K., and Engels, W. R. (1988) Genetics118, 461-470 [Abstract/Free Full Text]
  26. Fridell, Y.-W. C., and Searles, L. L. (1992) Mol. Cell. Biol.12, 4571-4577 [Abstract/Free Full Text]
  27. Yamaguchi, M., Nishida, Y., and Matsukage, A. (1995) Gene Exp.4, 183-193
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.


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