The glycogen phosphorylase-2 promoter binding protein in Dictyostelium is replication protein A

Article No. mb982239 J. Mol. Biol. (1998) 284, 903±913

The Glycogen Phosphorylase-2 Promoter BindingProtein in Dictyostelium is Replication Protein A

Xiao Wen, Pawjai Khampang and Charles L. Rutherford*

Biology Department, Molecularand Cellular Biology SectionVirginia Polytechnic Instituteand State UniversityBlacksburg VA, 24061, USA

E-mail address of the [email protected]

Abbreviations used: gp2, glycogeDdRPA, the Dictyostelium homologprotein A; DIF, differentiation induelectrophoretic mobility shift assaysynthase; RPA, replication proteinreplication protein A.

0022±2836/98/490903±11 $30.00/0

During Dictyostelium development, glycogen degradation is a crucialevent that provides glucose monomers used in the synthesis of the essen-tial structural components for cellular differentiation. The product of thedevelopmentally regulated glycogen phosphorylase-2 gene (gp2) catalyzesthe degradation. DNA-binding proteins were found to bind to a regulat-ory site of the gp2 gene in a stage-dependent pattern. Gel-shift analysis ofundifferentiated amoebae cell extract revealed a protein migrating at0.40 Rf, while 17 hour differentiated cell extract produced a speciesmigrating at 0.32 Rf. Both the 0.32 and 0.40 Rf proteins were puri®ed andfound to consist of three subunits of 18, 35 and 62 kDa (for 0.40 Rf) or81 kDa (for 0.32 Rf). Data base searches identi®ed the protein as the Dic-tyostelium homologue of replication protein A (DdRPA). Amino acidsequence analysis showed identity between the 62 and 81 kDa subunits.Incubation of cell-free extracts under appropriate conditions at low pH,resulted in conversion of the 81 kDa to the 62 kDa subunit. Northern blotanalysis revealed that the levels of expression of the large subunit ofDdRPA were constant throughout differentiation and the size of themRNA was the same at all stages of development. The results raise thepossibility that pH induced post-translational modi®cations of DdRPA areinvolved in events that halt cell proliferation and induce differentiation inDictyostelium.

# 1998 Academic Press

Keywords: Dictyostelium; replication protein A; transcription factor;development
*Corresponding author
Introduction

One of the initial events that occurs during celldifferentiation of Dictyostelium is the coordinatedbreakdown of endogenous stores of protein andRNA (Franke et al., 1973; White et al., 1961; Wrightet al., 1959, 1960). The breakdown products arethen shunted into carbohydrate and temporarilystored as glycogen. As differentiation commences,glycogen degradation results in the formation ofglucose units that are subsequently used for syn-thesis of the carbohydrate structural componentsin the terminally differentiated state (Gustafsonet al., 1972; Marshall et al., 1970). In our laboratory,

ing author:

n phosphorylase-2;ue of replicationcing factor; EMSA,; gs, glycogenA; huRPA, human

the process has been studied extensively and genesfor two forms of glycogen phosphorylase (gp1 andgp2) and glycogen synthase (gs) have been clonedand characterized (Brickey et al., 1990; Narananet al., 1988a,b; Rutherford & Cloutier, 1986;Rutherford et al., 1997). During cell differentiation,the GP2 enzyme activity ®rst appears in prestalkcells where the degradation products are used forstalk construction. A few hours later, the presporemass is lifted off the substratum by the lengtheningstalk, and only then does gp2 become active in theprespore cells. As the prespore mass reaches theapex of the stalk, GP2 shows maximal activity, gly-cogen is degraded, and the synthesis of spore wallcellulose is completed (Rutherford, 1976;Rutherford & Cloutier, 1986).

Typically, the regulation of gene expression isexerted chie¯y at the level of transcription; there-fore, the major thrust of our investigation has beenthe analysis of transcription of these genes. Duringdevelopment, gp2 is regulated independently inthe two cell types by a sophisticated interplaybetween morphogens (Yin et al., 1994). Four diffu-

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904 The gp2 Promoter Binds Replication Protein A

sible molecules, cAMP, NH4�, adenosine, and

differentiation inducing factor (DIF), have beensuggested to function as cell-speci®c induction sig-nals (Berks et al., 1991; Kay et al., 1989; Kimmel &Firtel, 1991; McRobbie et al., 1988). The effect ofthese morphogens on gp2 gene expression wastested and the results showed the gp2 gene can beinduced by both DIF and cAMP, and that NH4

�

and adenosine modulate this regulation (Yin et al.,1994).

In order to de®ne the cis-acting elements thatregulate gp2 expression, a gp2-luciferase reportergene was constructed and found to accuratelyreport the expression of the endogenous gp2 geneduring development (Rutherford et al., 1997). Tode®ne which region(s) in the 50 untranslatedsequence of the gp2 promoter is involved in confer-ring optimal and temporal expression of the gp2gene, 50 deletions in the upstream sequences wereconstructed. A fragment from ÿ1216 to ÿ486 wasfound to be required for optimal expression, andthe region between ÿ486 to ÿ242 was required forbasal activity.

The effects of disrupting regulatory sequenceswith the region between ÿ379 to ÿ327 were inves-tigated using internal deletions and site-directedmutations (Wu, 1995). In the construction of thesemutations the upstream regulatory elements werekept intact. Mutation of this region resulted in upto 50-fold decrease in the developmentally inducedluciferase reporter gene activity.

Using an electrophoretic mobility shift assay(EMSA), with probes that were identi®ed as puta-tive cis-responsive elements by site-directed muta-genesis, we found stage-speci®c binding ofnuclear-enriched proteins (Rutherford et al., 1997).A change in the EMSA banding pattern occurredjust prior to activation of the gp2 gene. A band waspresent at 0.40 Rf with extracts from proliferatingcells and at one hour and four hours of differen-tiation. With extracts from cells taken 12 hoursafter plating, the 0.40 band was replaced with anew band at 0.32 Rf.

The speci®city of the interaction with the DNA-binding protein(s) was assessed by inclusion ofthe non-speci®c competitors poly(dI)-poly(dT),poly(dA)-poly(dT) or calf thymus DNA (0.1 to1 mg) and by competition analysis with a varietyof unlabeled oligonucleotides. No competition wasobserved with 133-fold excess of oligonucleotidesconstructed from an 82-mer EBNA-1 binding-site, a63-mer Oct-1 binding site, or ÿ596 to ÿ570 of thegp2 50 untranslated region. Although oligonucleo-tides containing the wild-type sequence competedfor binding at only tenfold excess over the labeledprobe, those containing the mutated sequences asused in the site-directed mutagenasis showedlower levels of competition. Likewise, if themutated sequences were labeled and used asprobes in the EMSA the 0.32 and 0.40 bands wereeither reduced or not present (Khampang, 1995;Wu, 1995; I. McCaffery, unpublished observation).Thus, point mutations in the regulatory element

reduce gene expression in vivo, and also reducedthe binding af®nity to the elements in the in vitroEMSA assay.

The DNA-binding activity was partially puri®edusing hydroxyapatite, DEAE 40 HR, and DNA af®-nity chromatography. The af®nity matrix consistedof the sequence from ÿ317 to ÿ347 tailed withpoly(A) nucleotides and coupled to oligo(dT) cellu-lose. However, the DNA-binding activity of theaf®nity-puri®ed fraction was unstable and, there-fore, precluded puri®cation of the protein(Rutherford et al., 1997).

Here, we have modi®ed the puri®cation schemeto include construction of an af®nity column froma concatenated version of the ÿ317 to ÿ347 bpdouble-stranded oligonucleotide, coupled to CNBr-Sepharose-4B. This modi®cation of the puri®cationprocedure provided greater capacity at the af®nitystep and, therefore, increased concentration of theeluted protein. This, coupled with a shortened timerequirement for the total puri®cation, led to thesuccessful puri®cation of the protein. Using thisprocedure, the DNA-binding protein was puri®edand was shown to consist of three subunits. Thesubunits were subjected to protein sequence anal-ysis and the large subunit was cloned and substan-tially characterized. The deduced protein sequencewas similar to that of the large subunit of replica-tion protein A (RPA). RPA has been shown tofunction in DNA replication, DNA repair, and as atranscription factor (Brill et al., 1989; Dutta et al.,1993; He et al., 1993; Lee et al., 1995; Li & Botchan,1993; Luche et al., 1993; Matsuda et al., 1995;Melendy & Stillman, 1993). It binds DNA as a het-erotrimer consisting of three subunits of approxi-mately 70, 34 and 12 kDa molecular mass. Thisreport is the ®rst time that replication protein Ahas been described in Dictyostelium. The resultssuggest a dual role for the protein in Dictyostelium,as a replication factor during proliferation of undif-ferentiated cells and as a transcription factorduring cell differentiation.

Results

pH-Dependent ``conversion'' between the0.32 Rf and 0.40 Rf band

We have shown previously that a change in theEMSA banding pattern occurs just prior to acti-vation the gp2 gene (Rutherford et al., 1997).A band with mobility of 0.40 Rf was alwaysobserved with extracts from proliferating cells andat one hour and four hours of differentiation. How-ever, with extracts from 12 hour cells, a new bandat 0.32 Rf replaced the 0.40 band. In the initialattempts to purify the protein(s) that bind to thegp2 promoter, the effectiveness of several chroma-tography resins were tested, including hydroxya-patite and CM-Sephadex. Because the capacity ofthese resins is greater at lower pH, some of theseanalyses were performed at pH 6.0 to 6.5. In someexperiments, the extract from differentiated cells

Figure 1. pH dependent ``conversion'' between the 0.32 Rf and 0.40 Rf band. A, EMSA of 17 hour cytosolic cellextracts assayed in different buffers with a range of pH. The reaction buffers in which the assays were performed areshown above the lane numbers. Lanes 1 and 2 show assays in Tris-HCl (pH 7.9) and Hepes reaction buffer (pH 7.9),respectively. Lanes 3, 7 and 11 were performed at pH 6.0, lanes 4, 8 and 12 at pH 6.5, lanes 5, 9 and 13 at pH 7.0,and lanes 6, 10 and 14 at pH 7.5. Numbers to the right are Rf. B, EMSA of cytosolic and nuclear extracts performedin different buffer conditions. The reaction buffers, Hepes (pH 7.9), K2HPO4/KH2PO4 (pH 7.4), and Mes (pH 6.3) areindicated above the lane numbers. Lanes 1, 5, 9 and 3, 7, 11 are cytosolic extracts from amoebae and 17 hour cells,respectively. Lane 2, 6, 10 and 4, 8, 12 are nuclear extracts from amoebae and 17 hour cells. C, Test of the reversibilityof the conversioni from 0.32 to 0.40 Rf. The hydroxyapatite fraction that contained 0.32 Rf activity was used in theassay from lane 1 to 8. A 17 hour cytoplasmic fraction was used in the assays from lane 9 to 18. The ÿ and � symbolrefers to the presence (�) of reaction buffer in which the assay was performed (Tris-HCl (pH 7.9) or Mes (pH 6.3)).Lanes 1, 2, 9, 10, 17 and 18 were assayed without further treatment of the samples. One aliquot of each sample waspassed though a Bio-spin 6 column containing 20 mM Mes buffer (pH 6.3), to lower the pH and allow conversion tooccur. This sample was assayed by EMSA in Tris-HCl or Mes reaction buffer as shown in lanes 3, 4, 11 and 12.Another aliquot of each extract was passed over an identical spin column equilibrated in 20 mM Tris-HCl buffer(pH 7.9) and then assayed by EMSA as shown in lanes 5, 6, 13 and 14. A sample of the elute from the ®rst Mes spincolumn was then passed over a second spin column that had been equilibrated in 20 mM Tris-HCl buffer (pH 7.9),then assayed in lanes 7, 8, 15, and 16. All samples were preincubated in the reaction mixture for 30 minutes beforeloading on the gel, except in lanes 17 and 18 where there was no preincubation before loading on the gel. Lanes 19and 20 are the same as lanes 7 and 15, respectively.

The gp2 Promoter Binds Replication Protein A 905

was prepared in Tris-HCl buffer at pH 7.9 andthen applied to a column that had been equili-brated at pH 6.0 to 6.5. In this case, the sampleapplied to the column revealed an EMSA band at0.32 Rf (characteristic of the differentiated cells),while the fractions eluting from the column con-tained primarily the 0.40 Rf band (found only inundifferentiated cells, data not shown). If, how-ever, the chromatography was performed entirelyin buffer at pH 7.9, the 0.32 Rf band was retainedin the eluted fractions. This result suggestedthe possibility of a pH dependent ``conversion''between the 0.32 and 0.40 Rf bands.

To test this, the DNA binding activity in 17 hourcytosolic cell extracts was analyzed by the EMSAin several different buffers over a range of pH. AtpH 7.0 or 7.5, retarded bands appeared at 0.22 and0.32 Rf (Figure 1A, lanes 5, 6, 9, 10, 13 and 14). AtpH 6.5, a retarded band located at 0.40 Rf wasobserved, corresponding to diminished intensity ofthe 0.22 and 0.32 Rf bands (lanes 4, 8 and 12). Ifthe pH was decreased to 6.0, the EMSA showednearly complete loss of the 0.32 band and a corre-sponding increase in the intensity of the 0.40 Rfband (lanes 3, 7 and 11). The same result wasobserved for Mes, K2HPO4/KH2PO4, and imida-zole buffers.

We also tested the effect of pH on the migrationof the retarded bands in both cytoplasmic andnuclear fractions (Figure 1B). With extracts fromthe amoebae stage, no effect of pH was found on

either the nuclear (lanes 2, 6 and 10) or cytoplasmicfractions (lanes 1, 5 and 9). In each case, the charac-teristic 0.40 Rf band was present. With 17 hourcytoplasmic extracts, EMSA at pH 6.3 resulted inthe loss of the 0.32 Rf band, and appearance of the0.40 Rf band (compare lanes 3, 7 and 11). How-ever, with 17 hour nuclear extracts, there was noconversion of the 0.32 to 0.40 Rf band (comparelanes 4, 8 and 12). Thus, at low pH, EMSA of 17hour nuclear extract showed no in vitro conversionof the 0.32 Rf band to 0.40 Rf, while in cytoplasmicextracts of the 17 hour cells the conversion wasreadily apparent.

We also tested if the conversion of the 0.32 Rfband to the 0.40 Rf band in the 17 hour cytosolicextract was reversible. Two samples were used: anunfractionated 17 hour cell extract in 20 mM Tris-HCl (pH 7.9; Figure 1C, lanes 9 to 18), and a par-tially puri®ed preparation from hydroxyapatitechromatography in which the active 0.32 Rf bandwas eluted at approximately 300 mM K2HPO4/KH2PO4 (pH 7.9; Figure 1C, lanes 1 to 8). The lattersample was maintained in the high concentrationbuffer so as to retain the high pH condition in sub-sequent incubations. EMSA of the two samplespreincubated in Mes (pH 6.3) or Tris-HCl (pH 7.9)reaction mixture showed the usual conversionfrom 0.32 to 0.40 Rf bands with the extract in20 mM Tris-HCl (pH 7.9; Figure 1C, lanes 9 and10), but no conversion with the extract in 300 mMphosphate buffer (pH 7.9; lanes 1 and 2). The lack

Figure 2. EMSA of the DEAE-Sephacel batch chroma-tography, with extracts from six hour and 17 hour cells.A, The six hour cell extract. The 0.4 Rf band activity inlane 1 was from the sample that was applied to theresin. Flow-through fractions are shown in lane 2, (FT-1)and lane 3 (FT-2). Lanes 4, 5, and 6 are the 300 mMNaCl elute fraction-1, fraction-2, and the 1 M NaCl elutefraction, respectively. B, The same chromatography wasperformed using the 17 hour cell extract. Lane 1 showedthe 0.32 Rf band activity in the sample that was appliedto the resin. The assay of the ¯ow-through fractions isshown in lanes 2 and 3. The protein eluted from theresin at 300 mM NaCl is shown in lane 4 (fraction-1)and in lane 5 (fraction-2). The 1 M NaCl fraction wasnot included in this assay.


of conversion of the hydroxyapatite fraction wasdue to the 300 mM phosphate buffer (pH 7.9) thatwas carried into the reaction mixture with thesample, thus maintaining the high pH. The pH ofboth the hydroxyapatite fraction and the sample in20 mM Tris-HCl (pH 7.9) was then lowered bypassing them through a Bio-spin 6 (Bio-Rad) col-umn that was pre-equilibrated in 20 mM Mes buf-fer (pH 6.3). As a control, another aliquot of eachextract was passed over identical spin columnsequilibrated in 20 mM Tris-HCl (pH 7.9). Extractsthat had passed through the low pH Mes spin col-umn and then preincubated in either the high pHTris-HCl buffer or in the low pH Mes buffershowed only the 0.40 Rf band (Figure 1C, lanes 3,4, 11 and 12). Thus, once the sample was placed inlow pH and had converted to the 0.40 Rf band, itcould not shift back to the 0.32 Rf position evenwhen incubated in the high pH reaction buffer.The control extracts that were passed over the spincolumn equilibrated in Tris-HCl (pH 7.9), showedthe same pattern as extract that was not subjectedto spin column treatment; that is, the appearanceof the 0.40 Rf band occurred only in the low pHMes reaction buffer (lanes 5, 6, 13 and 14). Thisresult shows that there was no effect on the extractby simply passing it over the spin column. To testfurther if the 0.40 Rf band could convert to the0.32 Rf band, the elute from the Mes spin columnwas passed over a second spin column equilibratedin 20 mM Tris-HCl (pH 7.9). When this samplewas assayed by the EMSA, no conversion back tothe 0.32 Rf form was observed (lanes 7, 8, 15 and

16). We conclude that once the protein is convertedfrom the 0.32 to the 0.40 Rf form, it cannot returnto the 0.32 Rf by restoring the high pH environ-ment. In addition, no loss of the 0.32 Rf band wasobserved if the sample in high pH Tris-HCl bufferwas placed in low pH Mes reaction mixture andthen immediately loaded on the gel without pre-incubation (the zero time control; lanes 17 and 18).Thus, the conversion reaction is time-dependent.

We also tested the possibility that the 0.32 Rfform of the binding protein was present in amoe-bae extracts but was cleaved to the 0.40 band afterthe cells were lysed. Amoebae were lysed by a var-iety of methods, some of which are known torelease proteases from lysosomes and membranevesicles, and others that retained the organelles.The extracts were then immediately assayed by thegel shift analysis. With all methods of cell ruptureonly the 0.40 band was observed. In addition, onlythe 0.40 band was observed when cells were lysedin the presence or absence of the protease inhibi-tors PMSF, TAME, TLCK, TPCK, EDTA, benzami-dine, pepstatin A, leupeptin, chymostatin, antipain,phenanthroline and aprotinin. In fact, we havenever observed the 0.32 Rf band in amoebaeextracts under any of the many conditions we haveused to study the protein. Intermediate bandsbetween 0.32 and 0.40 Rf were not observed in anycase, nor was further degradation of the 0.40 bandobserved in extended incubations of the crudeextracts, or during the several days that wererequired for puri®cation of the protein. The resultssuggest that the 0.32 to 0.40 conversion was notdue to random proteolytic activity in vitro.

Purification of the DNA binding protein(s)

The extract was ®rst subjected to column chro-matography using DEAE-Sephacel and the DNA-binding activity in each fraction was determinedby EMSA. If undifferentiated cell extract was used,a 0.40 Rf band was retained by the resin andeluted in the ®rst 300 mM NaCl fraction (300-1;Figure 2A, lane 4). If the 17 hour cytosolic extractwas used, the resulting 0.32 Rf band was alsoeluted in the 300 mM NaCl fraction (Figure 2B,lane 4). These fractions were then subjected toDNA-af®nity chromatography as described inMaterials and Methods. Analysis of the af®nityfractions by EMSA revealed that the DNA-bindingactivity eluted at approximately 0.7 M NaCl witheither six hour or 17 hour extracts (Figure 3A,lanes 4, 5 and 6, and Figure 3B, lanes 4, 5 and 6,respectively).

SDS-PAGE analysis of the af®nity puri®edsamples from six hour extracts revealed three pep-tides of 62, 35, and 18 kDa in fractions that con-tained DNA binding activity (Figure 4A, lanes 6, 7and 8). If the 17 hour extract was used, SDS-PAGEseparation showed three bands at 81, 35, and18 kDa (Figure 4B, lanes 4, 5 and 6). In 12 separatepuri®cations of the gp2 promoter binding proteins,the fractions containing these bands always corre-

Figure 3. EMSA of fractions from the DNA af®nitychromatography, with extracts from six hour and 17hour cells. A, The 300 mM fractions from DEAE-Seph-acel chromatography of six hour cell extract was appliedto the af®nity column. Lanes 1 to 10 are the fractionscollected during the salt gradient. The DNA-bindingprotein eluted from the column at about 700 mM NaCl,as shown by the 0.40 Rf band in lanes 4 and 5. B, Thesame chromatography was performed with the 17 hourcell extract. The gradient fractions are lanes 1 to 9. The0.32 Rf band activity also eluted at about 700 mM NaClconcentration as shown in lanes 4 and 5.


sponded to fractions showing DNA bindingactivity. Additionally, EMSA of 0 to six hourextracts or 17 hour extracts always resulted in 0.40and 0.32 Rf bands, respectively. Thus, the 62 and

Figure 4. SDS-PAGE of the DNA af®nity fractions.A, A 1 ml sample was taken from each of the 10 ml sixhour af®nity fractions, TCA-precipitated, then analyzedby SDS-PAGE. The gel was stained with Brilliant BlueG. Lanes 1 and 2 are from af®nity ¯ow-through 1 and¯ow-through 2 fractions; Lanes 3 to 12 are the elutedfractions corresponding to lanes 1 to 10 of Figure 3A.Three peptides of 62, 35 and 18 kDa were present inlanes 6, 7 and 8. Their relative sizes were determined bycomparison to the protein standard marker in lane 13.B, SDS-PAGE of the 17 hour DNA af®nity chromatog-raphy. The same experiment as in A was performed.The lane numbers correspond to those shown inFigure 3B. An 81 kDa band was present in the samefractions as the 35 and 18 kDa peptides, lanes 4, 5 and6. Lane 10 contains a molecular mass marker.

Figure 5. Gel ®ltration chromatography of active frac-tions from the DNA af®nity chromatography. The activefractions from the af®nity column were concentrated to500 ml and applied to a 300 SW gel ®ltration column(0.8 cm � 30 cm; Waters) in Tris-HCl buffer (pH 7.9)containing 300 mM NaCl. The ¯ow rate was 0.5 ml/min. The resulting fractions were analyzed by SDS-PAGE (A), and EMSA (B).

81 kDa peptides appeared to be related to themigration of the retarded bands in the EMSA of0.40 and 0.32 Rf. In addition, both the EMSA mobi-lity and the size of the large subunit showed thesame temporal expression during development.

In order to test further if the three peptides thatco-eluted from the DNA af®nity column formed aheterotrimer, we performed gel ®ltration chroma-tography (300 SW, Waters) in Tris-HCl buffer(pH 7.9) containing 300 mM NaCl (Figure 5).Under these conditions, the 62(81), 35, and 18 kDasubunits eluted in the same fraction (Figure 5A). Inaddition, the DNA-binding activity co-eluted withthe three peptides (Figure 5B). We conclude thatthe gp2 promoter binding protein is a functionalheterotrimer.

Southwestern blot analysis

To determine which of the subunits containedDNA-binding properties, Southwestern blot anal-ysis was performed using the af®nity fractions

Figure 6. Autoradiograph of the Southwestern blotanalysis of the puri®ed protein from the 17 hour cellextract. The puri®ed protein was blotted onto a nitrocel-lulose membrane, then probed with 32P-labeled oligonu-cleotide. The autoradiograph showed that the 81 kDasubunit contained the DNA-binding activity.


from 17 hour extracts. The pooled af®nity fractionscontaining DNA-binding activity were precipi-tated, separated by SDS-PAGE, and transferredonto a nitrocellulose membrane. The membranewas then probed with the labeled ÿ322 to ÿ346 bpoligonucleotide both in the presence and theabsence of non-speci®c poly(dI)-poly(dC). Auto-radiography of the hybridized membrane (Figure 6)showed that only the 81 kDa subunit bound to theDNA probe.

Amino acid sequencing

In order to obtain a suf®cient amount of the pur-i®ed protein for microsequencing, fractions con-taining DNA binding activity from ten separateaf®nity columns were pooled. We ®rst attemptedto sequence the intact peptides, but all three werefound to be blocked at the amino terminus. There-fore, each peptide band was subjected to proteo-lytic cleavage, separated by SDS-PAGE, and thentransferred onto PVDF membrane for amino acidsequencing (see Materials and Methods). Identicalsequences were observed between two fragmentsresulting from V8 digestion of the 81 kDa and62 kDa peptides (data not shown).

Cloning of the large subunit

In order to obtain a probe with which to screena library for the gene encoding the large subunit,

several pairs of degenerate primers were designedand used for PCR ampli®cation of the gene(s)using genomic DNA template. A fragment of800 bp was ampli®ed by touchdown PCR. ThisPCR fragment was cloned, sequenced, and foundto contain a single EcoRI site. To con®rm that thePCR product was a true copy of the gene encodingthe 62 kDa (81 kDa) subunit, we compared thederived amino acid sequence and the peptidesequence. Not only were the PCR primersequences found, but the derived amino acidsequence 30 to the primers was found to be identi-cal with that of the amino acid sequence of thepuri®ed protein.

The 800 bp PCR product was 32P-labeled, thenused as a probe to screen a partial EcoRI genomicDNA library. Although the 50 untranslatedsequence and the N-terminal coding region of thegene encoding the large subunit was obtained, theremainder of the 30 region of the gene was notfound in the EcoRI genomic DNA partial library.A HindIII genomic DNA sub-library was thenconstructed as described in Materials and Methodsand screened with the 800 bp PCR product.Sequencing of the resulting clones produced sev-eral fragments that matched amino acid sequencesobtained from the protein, thus con®rming theiridentity. In addition, PCR ampli®cation wasattempted using one primer from the 81 kDa sub-unit sequence and one from the 62 kDa sequence.This resulted in a single fragment. Sequence com-parisons of this product with those obtained fromvarious combinations of primers from the 62 and81 kDa subunits showed complete identity. Like-wise, extensive genomic Southern analysis using62 and 81 kDa probes showed identical patterns(data not shown). Thus, we conclude that the 62and 81 kDa peptides are products of the samegene.

Search of sequence databases

A search of sequence databases revealed that theonly sequence with high similarity was that ofreplication protein A (RPA), a DNA replication fac-tor. Further analysis showed that the 62 kDa(81 kDa) subunit had high similarity (54.9%) withthe large subunit of human replication protein A(huRPA70; Figure 7). The central part of thesequence was more conserved than the N-terminalor the C-terminal region. RPA is a heterotrimericDNA binding protein that has been identi®ed inseveral eukaryotic systems (Brown et al., 1992; Kimet al., 1992; Umbricht et al., 1993). In addition tobeing required for DNA replication (both initiationand elongation), RPA is also involved with in vivoDNA repair and recombination and gene regu-lation (Longhese et al., 1994). The structure of theheterotrimeric protein is highly conserved amongspecies, consisting of three polypeptide subunitswith approximately 70 kDa, 34 kDa, and 12 kDamolecular mass. The DNA-binding activity residesin the 70 kDa subunit. Thus, the subunit structure,

Figure 7. Comparison of the pro-tein sequence between DdRPA1and the human RPA large subunit.

Figure 8. Northern analysis of the expression level ofthe DdRPA1 mRNA during development. Four micro-grams of total RNA puri®ed from different stages ofdevelopment were used in each lane: lane 1 and 2, zerohour; lane 3 to 7, 4, 8, 12, 16 and 20 hours, respectively.The RNA bands were quantitated by optical densitome-try (NIH Image). A, DdRPA1 PCR fragment was theprobe. B, (as a standard) The Northern blot membraneused in A was stripped and then reprobed with an actin8 PCR fragment.


the DNA-binding domain and the amino acidsequence are similar to the protein that was puri-®ed and, therefore, we conclude that it is the Dic-tyostelium homologue of RPA. We call this proteinDdRPA and the 81 (62), 35, and 18 kDa subunitsDdRPA1, DdRPA2, and DdRPA3, respectively.

Primer extension to determine the start siteof transcription

To locate the transcription start site, total RNAfrom both undifferentiated and differentiated cells(slug stage) was used as a template for the reversetranscriptase reaction. Primer extension from twoseparate sequences produced a single band at aposition corresponding to 306 bp upstream of the®rst ATG. The same result was obtained whetherthe RNA template was from the differentiated orundifferentiated cells (data not shown). It is likely,therefore, that the 81 to 62 kDa transition is at thepost-translational level rather than through the useof alternate promoters.

Northern blot analysis of the 62 kDa(81 kDa) gene

To determine the size of the DdRPA mRNA andits developmental expression, RNA was puri®edfrom different developmental time points and sub-jected to Northern blot analysis (Figure 8A). As acontrol, the same membrane was probed using aDictyostelium actin 8 gene fragment (Figure 8B).It is known that the expression level of actin8 mRNA is constant throughout development,then decreases dramatically at 20 hours (Romanset al., 1985). The level of 12 hour actin 8 mRNAwas set as a standard, and the readings in othertime points were compared with it. Variation inthe level of actin 8 mRNA was taken as experimen-tal error and then used to correct the level ofDdRPA1 mRNA. The results show that the mRNAof the large subunit was expressed at a constantlevel during development.

The size of the mRNA coding for this subunit,approximately 2600 nt, is the same at all stages ofdevelopment, despite the fact that the size of thepeptide increases from 62 to 81 kDa during celldifferentiation. This is additional evidence that thelarge subunit of DdRNA is modi®ed at the post-translational level. The size of the mRNA also indi-cates that approximately 340 bp are represented bynon-translated sequence.

Discussion

Since it was ®rst described only nine years ago,RPA has received the attention of investigatorsfrom several diverse research areas. This wide-spread interest in the molecule stems from its mul-tifunctional role in the cell, including: (1) DNAreplication; (2) DNA repair and recombination; (3)


interaction with tumor suppressor gene products;and (4) regulation of transcription (Brill et al., 1989;Dutta et al., 1993; He et al., 1993; Lee et al., 1995; Li& Botchan, 1993; Luche et al., 1993; Matsuda et al.,1995; Melendy & Stillman, 1993). RPA has beenidenti®ed in several eukaryotic systems includinghuman, calf thymus, Xenopus laevis, Drosophila,Saccharomyces cerevisiae and a trypanosomatid(Crithidia fasciculata; Brown et al., 1992; Umbrichtet al., 1993). The subunit structure is highly con-served among species, consisting of three poly-peptide subunits with approximately 70 kDa,34 kDa, and 12 kDa molecular mass. The multi-subunit structure may be required to makeprotein-protein contacts because heterologoussingle-stranded binding proteins cannot substitutefor RPA in replication or repair (He et al., 1995).RPA is known to interact with several transcrip-tion factors, including, VP16, GAL4, XPA (theproduct of the gene defect in Xeroderma pigmento-sum), and the tumor suppressor gene product,p53 (Dutta et al., 1993; Li et al., 1995; Singh &Samson, 1995). It has recently been demonstratedthat RPA binds the product of the tumor sup-pressor gene, p53, with high speci®city (Duttaet al., 1993; Li & Botchan, 1993). This interactioninhibits the ability of RPA to bind single-stranded DNA, suggesting that the suppressoraction of p53 might include sequestration ofRPA from the rest of the replication machinery.Yeast RPA also binds directly to upstreamelements that mediate negative and positive con-trol of the transcription of several genes (Lucheet al., 1993). The DNA-binding activity resides inthe 70 kDa subunit, and its gene encodes a zinc®nger motif (Murti et al., 1996; Wold & Kelly,1988). The 30 kDa subunit can be multi-phos-phorylated, causing a characteristic shift in mobi-lity upon SDS-PAGE. Phosphorylation can beinduced in vivo by UV or X-ray DNA damage, aresult that undoubtedly re¯ects RPA's role inDNA repair. During the cell cycle, RPA is phos-phorylated at the G1 to S-phase transition andthen dephosphorylated at mitosis, thereby reset-ting the cycle (Fotedar et al., 1992). Cyclin-cdc2kinase can phosphorylate the same residues ofRPA in vitro as are phosphorylated in vivo,although additional sites are also phosphorylatedduring the cell cycle (Dutta & Stillman, 1992;Fang et al., 1993; Henricksen & Wold, 1994).Several phosphorylated forms are present inDrosophila and are tightly regulated at speci®cstages of cell differentiation (Mitsis, 1995). Therole of the 12 kDa subunit has not been deter-mined.

In view of what is know about RPA in the litera-ture and the information presented here, an intri-guing possibility is that recruitment of RPA tofunctions involving transcriptional regulationduring cell differentiation results in inhibition ofDNA replication and cell cycling. This modelrequires that RPA become targeted to sites of tran-scription, thus becoming unavailable for binding to

single-stranded DNA in the replication process.This could be brought about by post-translationalmodi®cation of the protein, thereby increasing af®-nity for promoter-binding sites active in transcrip-tion, and decreasing the af®nity for binding tosingle-stranded DNA required for DNA replica-tion. In this regard, we have found a characteristicchange in EMSA from 0.40 Rf to 0.32 Rf in prolifer-ating and differentiating cells, respectively. Inaddition, lowering the pH of extracts from differ-entiated cells resulted in loss of the EMSA band at0.32 Rf, and the appearance of the band that hadbeen observed only in undifferentiated cells (at0.40 Rf). We found that once the sample was con-verted to the 0.40 Rf band, it could not shift backto the 0.32 Rf position when returned to a high pHbuffer. In addition, at high pH the large subunitexisted as the 81 kDa form, but conversion to the62 kDa form was achieved by lowering the pHfrom 7.9 to 6.0 and incubating under the appropri-ate conditions. No change in size was observed forthe 35 and 18 kDa subunits.

Further evidence for post-translational modi®-cation of the DdRPA came from Northern analysisof the large subunit. No change was observed in:(1) the level of expression; (2) the size of themessage; or (3) the start site of transcription inundifferentiated cells and at all stages of cell differ-entiation. The size of the mRNA, 2600 bases, corre-sponded to a polypeptide of approximately81 kDa.

These results support the idea that the twoforms of DdRPA are the product of the same geneand are expressed in both proliferating and differ-entiating cells. The large subunit of DdRPA is syn-thesized as the 81 kDa polypeptide in bothproliferating cells and during cell differentiation,but is cleaved to the 62 kDa form in cells that areactively dividing. Lowering the pH in vitro resultsin conversion of the 81 kDa to 62 kDa form of thelarge subunit. This pH induced cleavage of thelarge subunit may also occur in vivo, for severallines of evidence show an increase of cytoplasmicalkalization occurs in Dictyostelium (Aerts, 1988;Aerts et al., 1985, 1986, 1987a,b; Edmonds et al.,1995; Gross et al., 1983; Jamieson et al., 1984; VanDuijn et al., 1991).

Based on the temporal expression of gp2, itsdemonstrated responsiveness to differentiation sig-nals, and the requirement of glycogen turnoverduring differentiation, we thought it likely thatanalysis of the control of this gene would uncoverbasic transcriptional determinants involved in thegenetic switch from cell proliferation to cell differ-entiation. However, we could not have predictedhow direct the link would be; that the proteinbinding a regulatory sequence of gp2 was, in fact,known also to be involved in the initial steps ofDNA replication. Thus, DdRPA may be representa-tive of the molecular components of the geneticswitch from proliferation to differentiation. Furtherstudies of DdRPA may uncover some of the detailsof one of the unifying principles of differentiation:


the molecular framework underlying a cell'sdecision to depart from the cell cycle and to enterinto a program of differentiation.

Materials and Methods

Cell culture and extract preparation

Dictyostelium discoideum strain AX3 K was grown inHL5 medium as described (Rogers et al., 1994). Cell-freeextracts were prepared by suspending cells in ®vevolumes of buffer A (0.5 mM EDTA, 150 mM sucrose,2% (v/v) Nonidet P40, 25 mM Tris-HCl, pH 7.9) for15 minutes at room temperature, then centrifugation at10,000 g for ten minutes. The supernatant was removedand stored at ÿ80�C. The pellet was suspended in twovolumes of the original cell pellet in buffer B (5 mMDTT, 5 mM Na2EDTA, 1 M NaCl, 5 mg/ml PMSF, 5 mg/ml leupeptin, 20 mM Tris-HCl (pH 7.9)), incubated onice for 30 minutes, then centrifuged at 150,000 g for60 minutes. The supernatant was removed and stored atÿ80�C.

To induce development, 108 cells were pelleted fromHL5 media by centrifugation at 1300 g for three minutes.The supernatant was discarded, the cell pellet waswashed twice with Milli Q water, then suspended inthree volumes of Mes-LPS buffer (20 mM KCl, 5 mMMgSO4 and 0.2 mM CaCl2 in 7 mM Mes buffer (pH 6.5)).The suspension was evenly spread on a 47 mm GN-6cellulose ester-based membrane (pore size 0.45 mm) sup-ported by a 47 mm absorbent cellulose pad soaked withMes-LPS buffer in a 6 cm diameter petri dish. The cellswere then incubated at 20�C for various time periods.

DEAE-Sephacel batch assay

The thawed cell extract was brought to 10 mg/ml withPMSF, then protamine sulfate was slowly added to a®nal concentration of 6 g/l of original packed cells. Afterstirring at 4�C for 15 minutes, the extract was centri-fuged at 20,000 g for 30 minutes. The supernatant wasremoved, passed through glass wool, added to DEAE-Sephacel resin (Pharmacia, 1 g resin per 6 ml packedcells) in a one liter bottle and shaken on the rotary sha-ker at 150 rpm for 60 minutes at 23�C. The resin waspoured into a column (5 cm � 6 cm) and washed with20 mM Tris-HCl buffer (pH 7.9) until the optical densityreached a basal level. Bound proteins were eluted fromthe resin with 20 mM Tris-HCl (pH 7.9) containing300 mM NaCl (¯ow rate 20 ml/min). This material (the300-1 fraction) was collected until the absorbance startedto decline, then another fraction was collected (the 300-2fraction) until the absorbance reached base line. Finally,20 mM Tris-HCl (pH 7.9) containing 1 M NaCl wasapplied to elute the remainder of the bound proteins.The active fraction (300-1) was brought to 0.4 M NaClbefore being stored at ÿ80�C.

Preparation and use of a DNA affinity column

The DNA af®nity column was prepared as describedby Kadonaga & Tjian (1986). An oligonucleotide withthe sequence 50 GATCCAGTGCAAACTCACCCACTCA-CAAT 30 and its complementary strand were annealed, 50phosphorylated, and ligated. The ligated oligomers werethen coupled to 5 g of CNBr-Sepharose-4B (Pharmacia),and stored at 4�C until required. The 300 mM elute fromthe DEAE Sephacel column (300-1) was thawed and

passed over the column at 2 ml/min. After the absor-bance had returned to base line, the bound proteins wereeluted for 45 minutes with a linear 0.3 M to 1.4 M NaClgradient in 20 mM Tris-HCl (pH 7.8) at the rate of 1 ml/min as formed by a Waters 650 protein puri®cation sys-tem. DNA-binding activity was determined by EMSA, asdescribed previously (Rutherford et al., 1997).

Preparation of peptides for microsequencing

The fractions showing DNA-binding activity from tenaf®nity steps were pooled, separated by SDS-12% PAGE,then stained with 0.1% (w/v) Brilliant Blue R. Peptideswere electroeluted into a Centricon 3 microconcentrationcell (Amicon) in 1.3 mM EDTA, 1.8% (v/v) glycine,0.05% (w/v) SDS and 25 mM Tris-HCl (pH 7.9), at 200 Vfor two hours. For cleavage of the eluted proteins, eithertrypsin or protease Staphylococcus aureus, strain V8, wasadded at a ratio of 1:40 (protease: target protein on aweight basis), then incubated overnight at 37�C. Thedigested products were separated in a 15% polyacryl-amide gel by SDS-PAGE and transferred onto a PVDFmembrane. After staining with 0.1% Brilliant blue, theprotein bands were excised and stored at ÿ20�C for pep-tide micro-sequencing (Matsudaira, 1993). The puri®edpeptides were sequenced using an Applied Biosystemsmodel 477A sequencer with on-line identi®cation of phe-nylthiohydantions at the protein sequencing facility atVirginia Polytechnic Institute and State University.

Touchdown PCR

The PCR reaction mixture contained 1 mg genomicDNA, 25 pmol of each degenerate primer, 10 nmol ofeach dNTP, 1.5 mM MgCl2, 50 mM KCl, in 10 mM Tris-HCl (pH 8.3). The reaction was heated at 75�C for twominutes, then 1 unit of Taq DNA polymerase was added.After denaturation at 94�C for two minutes, 12 cycles ofampli®cation were carried out, with denaturation at 94�Cfor 30 seconds, annealing at 56�C to 50�C for 30 secondsand extension at 72�C for 90 seconds. The annealing tem-perature was decreased 1�C every second cycle from 56�Cto a ``touchdown'' at 50�C. This was followed by 40additional cycles with the annealing temperature at 50�C,followed by a ®nal extension at 72�C for ten minutes.

Construction and screening of a sub-genomic DNAlibrary and an EcoRI partial genomic library

Ten micrograms of RNase-treated genomic DNA wasdigested overnight with 50 units of HindIII. After electro-phoresis on a 0.7% (w/v) agarose gel, the DNA frag-ments from 3 to 4 kb were separated into six fractions,then puri®ed by using a Geneclean Kit (Bio 101). PCRreactions were carried out to determine which fractioncontained the C-terminal region of the 62/81 kDa gene.The appropriate fraction was ligated to pBluescript vec-tor, and transformed into competent Escherichia coli(strain XL1-Blue). To identify positive colonies, PCRreactions on the resulting clones were carried out usingprimers speci®c to the DdRPA genes.

An EcoRI partial library was screened by hybridiz-ation at 65�C overnight with [a-32P]dATP-labeled PCRfragment probes (10 ng/ml of hybridization solution,approximately 108 CPM/mg), 1% (w/v) BSA, 1 mMEDTA, 7% (w/v) SDS and 200 mg/ml herring spermDNA in 0.5 mM NaHPO4 (pH 7.2) at 65�C. Positive colo-nies were picked from the original ®lters, grown on LB


plates overnight, then copied to ®lters. After a secondscreen, single colonies were picked, then grown on LB-ampicillin media. Following plasmid puri®cation, theDNA was subjected to Southern blot analysis andsequencing.

Southwestern blot analysis

The proteins were electroblotted onto a nitrocellulosemembrane as described above, then incubated in 50 mlBlotto (5% (w/v) non-fat powder milk, 50 mM NaCl,1 mM EDTA, 1 mM DTT, in 50 mM Tris-HCl (pH 7.5))and placed on a rocking platform at room temperaturefor 60 minutes. The membrane was washed three timesfor ®ve minutes each with 50 ml of binding buffer(50 mM NaCl, 1 mM EDTA, 1 mM DTT, in 10 mM Tris-HCl (pH 7.5)). The DNA-binding reaction was carriedout for three hours in 25 ml of binding buffer containing50,000 cpm/ml of 32P-labeled oligonucleotide and 1 mg/ml of non-speci®c competitor DNA (poly(dI)-poly(dC)).The membrane was washed four times for ®ve minuteswith 50 ml binding buffer, dried and subjected to auto-radiography at ÿ80�C.

Acknowledgments

We thank Laura Douglas for her excellent technicalassistance, and Dr Reyna Favis and Dr Ian McCaffery forproviding several of the probes and blots, for suggestingthe sub-genomic library and emphasizing the signi®-cance of the proliferation/differentiation transition, andfor reviewing the manuscript.

This research was supported by the National Institutesof Health grant AG 00678, and the Thomas F. Jeffressand Kate Miller Jeffress Memorial Trust grant J-222.

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Edited by M. Gottesman

(Received 17 March 1998; received in revised form 22 September 1998; accepted 22 September 1998)

The glycogen phosphorylase-2 promoter binding protein in Dictyostelium is replication protein A

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Transcript of The glycogen phosphorylase-2 promoter binding protein in Dictyostelium is replication protein A