Journal of Cell Science 102, 847-856 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

847

Molecular characterisation of the gene for the 180 kDa subunit of the DNA

polymerase-primase of Drosophila melanogaster

SIMON MELOV, HELEN VAUGHAN and SUE COTTERILL*

Department of Biochemistry, Imperial College of Science, Technology & Medicine, Exhibition Road, London SW7 2AZ, UK

•Author for correspondence

Summary

We have cloned and sequenced the gene for the 180 kDasubunit of the a polymerase from Drosophila melanogas-ter. The protein shows high similarity to the 180 kDasubunits from other species. Comparative expressionanalysis for the transcript, protein and enzymic activitysuggests that control occurs mainly at the level oftranscription. In situ analyses of the RNA suggest thathigh levels of the transcript are synthesised in the ovaries

and deposited uniformly in the egg. Immunolocalisationof the 180 kDa polypeptide in whole embryos shows thatits location is mainly nuclear; however, dispersal of theprotein can be seen to occur during mitotic phases of thecell cycle.

Key words: DNA polymerase-primase, Drosophila, DNAreplication.

Introduction

The DNA polymerase-primase a was the first polym-erase to be identified in eukaryotes (Bollum and Potter,1957), and for many years was thought to be the onlyreplicative polymerase. More recent evidence suggeststhat at least two additional polymerases (d and e) arenecessary to complete DNA replication. Although therelative contribution of each of these polymerases tothe synthesis of the leading and lagging strands is notunderstood, analysis of the SV40 in vitro systemsuggests that the role of the a polymerase is confined tosynthesis on the lagging strand, and possibly initiationat the origin of replication (Bambara and Jesse, 1991,for review). The <5 and e polymerases are not capable ofsubstituting for the a polymerase in this activity, sinceneither contains an associated primase activity.

Since its original isolation from calf thymus the apolymerase has been isolated from many differentorganisms and, across all species (from yeast to man),shows a remarkably conserved structure (Kaguni andLehman, 1988). The enzyme has four subunits; thelargest subunit (approximately 180 kDa) containspolymerase activity, the two smallest (50 and 60 kDa)constitute the primase, and a fourth (70-85 kDadepending on species) has no assigned function.Although these four subunits are normally tightlyassociated with each other, both polymerase andprimase activities are still efficient when the subunitsare separated from each other by means of denaturingagents (Cotterill et al. 1987a,b).

Recently, the cloning of various subunits of the a

polymerase has been reported from several species; thepolymerising subunit from Saccharomyces cerevisiae(Johnson et al. 1985; Lucchini et al. 1985), Schizosac-charomyces pombe (Damagnez et al. 1991), malarialparasite (R. Ridley, personal communication), andman (Wong et al. 1988); both primase subunits from S.cerevisiae (Lucchini et al. 1987; Foiani et al. 1989) andmouse (Prussak et al. 1989; Adler et al. 1991), and the73 kDa subunit from Drosophila (Cotterill et al.manuscript in preparation) and S. cerevisiae (Brooke etal. 1991). Analysis of these clones should aid in theunderstanding of the function of the a polymerase-primase in replication, and the role of the individualsubunits. It should also help to shed some light on themechanisms involved in control of the process ofreplication in eukaryotes, and its co-ordination withother cellular events.

We have initiated a project to clone all four subunitsof the a polymerase from Drosophila melanogaster.The Drosophila a polymerase is a well-characterisedenzyme (Kaguni and Lehman, 1988) and Drosophila iswell suited for a molecular biological approach to theanalysis of DNA replication: the amenability of theorganism to genetic study is well documented; inaddition patterns of development in Drosophila arehighly characterised, therefore facilitating an analysis ofthe interplay between this process and replication. Thispaper reports the isolation and characterisation of aclone for the 180 kDa subunit of the Drosophilapolymerase-primase. During the preparation of thispaper Hirose et al. (1991) also reported the isolation ofa clone for the same protein. The results presented here

848 S. Melov and others

show some significant differences from their data, andalso extend their observations on the expression of thesubunit both in vitro and in the whole organism bymeans of in situ hybridisation of mRNA and immuno-staining of the protein product.

Materials and methods

Bacterial strains and librariesDrosophila 0-4 h cDNA library in the vector pNB40 was a giftfrom Nick Brown. This library and all other transformationswere carried out using either DH5a- or TG2 as the host. Apreplated genomic library in Laurist 4 was a kind gift fromJorg Hoheisal. All cosmids were grown up in DH5a-. Cosmidnomenclature is as assigned by J. Hoheisel (personalcommunication).

DNA manipulationsAll restriction enzyme digests, ligations, end labelling,hexamer labelling, transformation, production of single-stranded phages (M13 and Bluescript (Stratagene)) andplasmids and genomic DNA was carried out as describedpreviously (Sambrook et al. 1990).

DNA blottingDNA samples were run through agarose gels of variouspercentages and transferred to Amersham Hi-bond N asdescribed previously (Sambrook et al. 1990). Prehybridisationand hybridisation conditions were 6xSSC (lxSSC is 0.15 Msodium chloride, 0.015 M trisodium citrate, pH 7), 5xDen-hardt's (lxDenhardt's is 0.02% BSA (bovine serum albu-men), 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 0.1% SDS(sodium dodecyl sulphate) and 100 /ig/ml salmon spermDNA, at 65°C. The filters were then washed in 2xSSC/0.1%SDS, 0.5xSSC/0.1% SDS, O.lxSSC/0.1% SDS, each for 15min at 65°C, and finally in 0.1 x SSQft. 1% SDS at 65°C for 1 h.

RNA isolation and characterisationTotal RNA was prepared by the method of Chomczynski andSacchi (1987). The RNA was separated by electrophoresisthrough 1.5% agarose gels containing 0.02 M MOPS (3-[N-morpholino] propane sulphonic acid), pH 5.5-7.0, 5 mMsodium acetate, 0.1 mM EDTA (ethylene diamine tetra-amino acid), 1.1% formamide. Before loading the gel theRNA samples were heated to 55°C for 10 min in 0.02 MMOPS, pH 5.5-7.0, 6.5% formamide, 0.5 mg/ml ethidiumbromide. The RNA was transferred onto a Hi-bond N filter asdescribed for DNA blotting except that no depurination ordenaturation steps were required. Prehybridisation andhybridisation were carried out in 0.4 M NaPO4, pH 7.2, 40%formamide, 4% SDS, 0.8 mM EDTA, 1 mg/ml BSA at 42°C.Washing of the filters was as described for DNA blotting.

DNA sequencingAll sequencing was carried out using the method of Sanger etal. (1977) on both single-stranded and double-stranded DNAtemplates. Most of the cDNA was sequenced by makingnested deletions in fragments of the clone in M13 vectorsusing a cyclone kit (International Biotechnologies Incorpor-ated, IBI), following the manufacturer's instructions. Forthose areas that were not covered by this method, specificoligonucleotide primers were synthesised and used as se-quencing primers on either double-stranded or single-

stranded DNA. All regions of the gene were sequenced onboth strands, and for most regions of the gene multiple clonesin each direction were analysed. Reconstruction of the finalsequence was carried out using the MicroGenie programmeson an IBM pc. Analysis of the protein and DNA sequenceswere carried out using the Daresbury system programmes.

Polymerase chain reaction (PCR)All PCR reactions were carried out for 30 cycles using thegeneral pattern of: denaturation for 1 min at 94°C, annealingfor 1 min at tx, and extension for 1 min at 72CC. The value oitxwas calculated independently for each set of primers used,and usually corresponded to the lowest tm of the two primersused for that particular run (calculated as 4xGC+2xAT).

Analysis of protein content and activityProtein samples from various stages of Drosophila wereprepared by homogenising the tissue in 15 mM Tris-HCl, pH8.0, 50 mM EDTA, 0.35 M sucrose, 0.5 mM DTT (dithioth-reitol), 2 mg/ml leupeptin, 1 mM PMSF (phenylmethylsulfo-nyl fluoride), 10 mM sodium bisulphite on ice. The homogen-ates were spun at 15,000 g for 20-30 min at 4°C, and the middleaqueous layer was retained. This was respun to remove thelast traces of cellular debris, and the samples were usedimmediately or stored at —70°C until required.

PAGE of protein samples was carried using the gel systemas described by Laemmli (1970).

The proteins were transferred to nitrocellulose by blottingfor 2-10 h in a Sartorius electroblotter. Blots were analysedusing both polyclonal and monoclonal antibodies.

Assays for DNA polymerase activity were carried out usingactivated calf thymus as the template as previously described(Hirose et al. 1988). Aphidicolin was added at a concentrationof 30 jig/ml.

In situ analysis of proteinsThe methods used for permeabilisation of embryos andhybridisation conditions were essentially as described pre-viously (Freeman et al. 1986). Where polyclonal antibodieswere used as the primary antibody, visualisation of the signalwas carried out using alkaline phosphatase-coupled secondaryantibodies. Experiments carried out with monoclonal anti-bodies (from G. Chui, unpublished data), were visualisedusing fluorescein-coupled secondary antibodies (Freeman etal. 1986).

In situ analysis of RNAPatterns of RNA expression in embryos and isolated ovariesof female Drosophila were analysed using dioxigenin-labelledRNA probes as previously decribed (Tautz and Pfeifle, 1989).

Results

Isolation of clones for the 180 kDa subunitDegenerate PCR primers were made for two regions ofamino acid sequence that were highly conservedbetween the published sequences of the yeast andhuman DNA polymerases (YGDTDS and TANSMY).These were used to amplify a 160 bp fragment fromDrosophila genomic DNA. This fragment was thenused as a probe to isolate 12 cDNA clones from a 0-4 hcDNA plasmid library and four genomic clones from agenomic library in Laurist 4.

Gene for 180 kDa subunit of DNA polymerase 849

positionso* Introna

genomlc

cONA

BX H E EX

BX H

Fig. 1. Comparative mapping of the cDNA with genomicclones. The position of the translational start site (ATG)and the approximate positions of introns are indicated. B,BamHl; E, EcoRl; H, HindUl; X, Xho\.

Analysis of the cDNA and genomic clonesRestriction analysis of clones

The cDNA clones were of various sizes, the longestbeing about 4.6 kb. There were three clones of this sizeand all showed identical restriction patterns. Compara-tive mapping of the cDNA and genomic clones wascarried out to locate the approximate positions ofintrons in the genomic DNA. A comparative map of thelongest cDNA and one of the genomic clones (15C11) isshown in Fig. 1. DNA blotting, using restrictionfragments from the cosmid clone to probe wild-typegenomic DNA was carried out to check that no DNArearrangements had occurred during cosmid cloning(data not shown).

Alignment of the restriction maps suggests that thechromosome of this gene contains at least four introns.The intron at the 5' end of the gene has been mappedprecisely by genomic sequencing (Fig. 1), but the exactarrangement of the other intron material has yet to bedetermined.

Quantitative genomic DNA blotting suggests thatthis is a single-copy gene, and in situ hybridisationshows that it maps to position 93F1 on the right arm ofchromosome 3 (data not shown).

Sequence of the cDNAThe nucleotide sequence of the cDNA clone, and theamino acid sequence associated with it is shown in Fig.2. Also shown on this diagram is a small section ofsequence from the upstream untranslated region of thegene, which was derived from the genomic clones. Theproposed transcriptional start site shows good corre-lation to the consensus for transcriptional starts inDrosophila (Hultmark et al. 1986), and at the 3' end ofthe gene there is homology to a recognised polyadenyl-ation signal, AUAAA (Biernstiel et al. 1985). Trans-lation of the protein from the presumed initiation codonwould produce a protein of 173 kDa.

Comparison with other known polymerasesThe 180 kDa subunit of the a polymerase fromDrosophila shows all six of the regions of homologypreviously described for eukaryotic polymerase cata-lytic subunits. It shows high similarity overall to the 180kDa subunits from both yeast (S. cerevisiae and 5.pombe) and man, although its relationship to the

human protein is slightly higher (37% identity ascompared with 27% for both yeasts). These homologiesoccur throughout the sequence, but are particularlyconcentrated at the 3' end of the gene (Fig. 3).

Developmental expression of the 180 kDa subunitDevelopmental expression of transcripts

The gene for the 180 kDa subunit directs the synthesisof a 4.8 kb message. Large amounts of the messagewere observed in ovaries and early embryos. The levelthen declines, and becomes undetectable at 16-20 h.The transcript remains undetectable at this sensitivitythroughout the rest of the life cycle (Fig. 4). These highlevels of maternal expression are consistent with thedemands for large amounts of DNA polymerasesduring the rapid cleavage divisions of the syncytialembryo. This pattern of expression has been observedfor many cell-cycle genes (Glover, 1991). In situhybridisation studies on ovaries show that the messagefor the gene first becomes visible in stage 2a, andremains visible up to the degeneration of the nurse cellsat stage 12. Entry of the RNA into the egg can be seenat stage 11. In situ analysis of embryos, at all stages,showed faint uniform staining at either high or lowmagnification (data not shown).

Developmental expression of the 180 kDapolypeptide

The expression profile for the 180 kDa protein, asdetermined by immunoblotting, is quite similar to thatfor the message (Fig. 5). Levels are high in earlyembryos, and then decline below the limits of detectionas the embryo ages further. For protein, however,additional peaks of expression occur during the firstinstar larval and early pupal stages. The variation inprotein also correlates well with patterns of aphidicolin-sensitive polymerase expression (Fig. 5). Since the apolymerase is thought to be the major constituent ofthis activity in embryos (Hirose et al. 1991), these datasuggest that there is no stockpiling of large amounts ofinactive polymerase.

Immunofluorescence staining has also been used tostudy the localisation of the 180 kDa polypeptide inovaries and embryos. All the staining shown here wasperformed with one monoclonal antibody. However,experiments performed with a different monoclonalantibody, and also polyclonal antibodies, gave similarresults. Immunofluorescence studies on ovaries (Fig.6D) revealed high levels of protein in the nuclei of thenurse cells and the follicle cells. For embryos thestaining observed is dependent on the particulardevelopmental stage (Fig. 7). In older embryos (cycle 7to approximately 80 min after the start of cycle 14)staining is clearly visible in the nucleus. Embryosyounger than cycle 6 show high levels of cytoplasmicstaining, although in some cases more intense stainingof nuclei is just visible. For stages later than 80 min afterthe start of cycle 14 the signal becomes too faint todetect at this level of resolution. For cycles 7-11 thestaining is uniform for nuclei in all regions of theembryo. However, in later cycles some areas of the

850 5. Melov and others

TTCCCGCCATOXaXTrTGATCATCCTCCAGCCCTGGTCACAAAACAATTTTTG^^

M S E S P S E P R A K R Q R V D K N G

TATTATCAAACTITAGCCAGTTXXJUVTGTCrGAATCACCGTCTGAACCGCGCGCTAAGCGCCAGAGAGTAGACAAAAATCS^

R F A A M E R L R Q L K G T K N K C K V E D Q V D D V

AGGTTCGCCGCCATGGAGCGACTGCGTCAGCTGAAGGGAACCAAGAACAAATGCAAAGTGGAGGACCAGGTGGACGATC3TA

Y D V V D E R E Y A K R A Q E K Y G D D W I E E D G T

G Y A E D L R D F F E D E D Y S D G E E D R K D L K

K K K G V A P N S K K R P R E N E K P V T G K A S I K

N L F S N A V P K K K M D V K T S V K D D D D I L A D

I L G E I K E E P A A T S E K A E K V I A P A K I S V

ATTTTGGGCGAAATAAAGGAAGAGCCTGCAGCGACGTCAGAAAAGGCGGAAAAGGTTATAGCCCCAGCGAAAATATCAGTT

T S R K F D A A A A K E Y M N S F L N N I K V Q E Q E

ACGTCGCGTAAATTO3ATGCCGCCGCTGCCAAGGAATACATGAATAGTTTTCTAAACAACATCAAAGTTCAGGAGCAGGAA

R K K A E A S S D N E M L E R I L K P K A A V P N T K

CGTAAGAAGCGGAGGCCAGTAGCGATAACGAGATGCTGGAGCGCATTCTGAAGCCCAAGGCAAGCAGTTCCAAACACAAAG

V A F F S S P T I K K E P M P E K T P A K K A T E D P

GTGGCCTTTITCTCCAGTCCTACAATCAAAAAAGAGCCCATGCCTGAAAAtyiCACCTGCAAAAAAAGCCACCGAAGATCCA

F S D N E M D F S C L D D D E N Q F D V E K T Q Q T E

TTCTCCGACAATGAAATGGACTTTAGCTGTCTGGATC^CGATGAAAACCAGTTTGATGTGGAGAAGACACAGCAGACCGAG

K V S Q T K T A A E K T S Q S K V A E K S A P K K E T

AAGGTTTCCCAAACGAAAACAGCAGCTCAGAAAACATCCCAAAGCAAGGTCGCCGAAAAATCAGCTCCCAAAAAAGAAACA

T G S P K E S E S E D I S R L L N N W E S I C Q M D D

ACTGGATCGCOlAAGGAATCGGAATCGGAGGACATT'AGTAGGTTGCTGAACAArrGGGAGTCTATATGCCAAATGGACGAT

D F E K S V L T T E Q D S T I S S D Q Q L R F W Y W E

GATITTX3AAAA<?raKTGCTCACCACGGAGCAGGArrCGACAATCTCCTCAGATCAACAGCTrcGGTTCTGGTACT^

A Y E D P V K M P G E V F L F G R T A D G K S V C L R

GCCTATGAGGATCCAGTAAAAATGCCCGGCGAAGTCTTCCTGTIXMGTCGGACCGCAGATGGAAAGTC^

V Q N I N R V L Y L L P R Q F L L D P I S K E P T K Q

GTACAGAACATCAACCGTGTGCTCTATCTGCTGCCCCGACAATTTCTCTTAGATCCCATTTCGAAGGAGCCCACCAA(5CAG

K V T V A D I Y K E F D S E V A N Q L K L E F F R S R

AAOJTCACCGTCGCAGACATATACAAGGAGTTCGACAGCGAGGTGGCAAACau^CTGAAGCTCGAGTTCTrTCGATCCCGC

K V T K S F A H H A I G I E V P Q S C D Y L E V H Y D

AAGGTCACCAAGAGTTTIXXXX^TCACGCTATCGGTATTGAAGTTCCGCAATCTTGCGATTACTTXX^GGTT^ACTACGAT

G K K P L P N L S A D K K Y N S I A H I F G A T T N A

L E R F L L D R K I K G P C W L Q V T G F K V S P T P

CTCGAAAGATTCCTCTTAGATCGGAAAATCAAAGGTCCCTGTTGGCTGCAGGTGACTG{^T^

M S W C N T E V T L T E P K N V E L V Q D K G K P A P

P P P L T L L S L N V R S M N P K T S R N E I C H T I

S M L T H N R F H I D R P A P Q P A F N R H M C A L T

TCCATGCTGACCCACAATCGTTTCCATATCGACCGCCCTGCTCCJIC^^

R P A V V S W P L D L N F E M A K Y K S T T V H K H D

CGCCCGtXMGTGGTTAGTTGGCCTITAGATTTGAACTTrGAGATGGCAAAA ATAAGTCTfvCCACAGTGCACAAACATGAC

S E R A L S V W F L A Q Y Q K I D A D L I V T F D S M

TCAGAGAGAGCCTTGTTGAGriCGTTCTTAGCTCAGTACCAGAAAATAGATGCAGATCTCATTGTAACCTTCGATTCCATG

D C Q L N V I T D Q I V A L K I P Q W S R M G R L R L

GATTGTCAACTAAATGTAATAACCGATCAAATTGTAGCCCTGAAGATTCCCCAATGGTCTCGAATGGGGCGACTGA<3GCTG

S Q S F G K R L L E H F V G R H V C D V K R S A E E C

AGCCAATCGTTTGGCAAGCGATTGCTGGAACATTTCGTCGGCAGAAT«rrTTGTGATGTAAAGCGATCTGC^

I R A R S Y D L Q T L C K Q V L K L K G S E R H E V N

ATAAGAGCAAGATCCTATGATCTGCAAACGCTATGCAAACAAGTACTGAAGCTTAA(5GGATC0aAGAGGATGGAAGTGAAT

A D D L L E H Y E K G E S I T K L I S L T M Q D N S Y

GCCGATGATCTGCTAGAGATCrrATGAGAAAGGAGAAAO^TTACCAAGCTCATATCGCTCACCATGCAGGACAATTCATAT

L L R L M C E L N I M P L A L Q I T N I C G N T M T R

T L Q G G R S E R N E F L L L H A F H E K N Y I V P D

ACCCTT^^AGGAGGTCGTTCTGAACGAAATGAGTTCTTGTTGCTGCATGCCTTCCACGAGAAGAATTAC^^

K K P V S K R S G A G D T D A T L S G A D A T M Q T K

AAGAAGCCGffTTTCTAAACGAAGTGGTGCJiGGAGATACGGATGCTACGCTTTCAGGTGCAGATGCGACGATGCAAACCAAA

K K A A Y A G G L V L E P M R G L Y E K Y V L L M D F

AAC»AGGCAGCCTATGCTGGT(XXriTAGTGTTGGAGCCCATGCGTG(?rCTCTACGAGAAGTACGTT^

N S L Y P S I I Q E Y N I C F T T V Q Q P V D A D E L

AACTCCCTGTACCCCAGTATAATACAGGAGTAC^ACATATGCTTCACCACCXnXrCAACAGCCXKTGGATGCAGATGAGT'^

P T L P D S K T E P G I L P L Q L K R L V E S R K E V

CCCACCCTACCCGACTCGAAAACGGAACCTGGAATCTTACCCCTTCAATTGAAACGCTTGGTGGA^

K K L M A A P D L S P E L Q M Q Y H I R Q H A L K L T

AAAAAGCTAATGG<^GCXCCTGATCTTrCGCCGGAACTCCAGATGCAGTACCACATTCGGCAGATGGaX^^

A N S M Y G C L G F A H S R F F A Q H L A A L V T H K

GCGAACTCCATGTATGGTTGCTTGGGrTTCGCCCACTCCO3GTrCTTTGCCC^GCACTTGGCnXr^^

G R E I L T N T Q Q L V Q K M N Y D V V Y G D T D S L

GGCAGAGAAATCCTCACGAACAC^CAACAACTCGTGCAAAAGATGAACTATGACGTTGTTTATGGAGACACAGATTCCCTG

M I N T N I T D Y D Q V Y K I G H N I K Q S V N K L Y

ATGATAAATACCAACATAACCGACTACGACCAGGTCTACAAGAtTGGTCACAATATTAAGCAGAGTGTAAATAAACTATAC

K Q L E L D I D G V F G C L L L L K K K K Y A A I K L

AAGCAGCTGGAATTGGATATCGATG<^GTATTTGGCTGTTTGCTCTTGCTGAAGAAAAAGAAGTATGCCGCTATCAAGTTG

S K D S K G N L R R E Q E H K G L D I V R R D W S Q L

AGCAAGG ATTCCAAGGGAAACCTGAGGCGTGAACAGAGCACAAGGGCCTGGATATAGTACGTCGCGATTGGTCTCAGCTG

A V M V G K A V L D E V L S E K P L E E K L D A V H A

GCTGTAATGGTGGGAAAAG(XGTTCrKX»TGAAGTGCTGTCGGAAAAGCCTTTGGAGGAGAAACTGGATGC^^

Q L E K I K T Q I A E G V V P L P L F V I T K Q L T R

CAACTGGAGAAGATCAAAACTC^AATTGCAGAGGGTGTAGTACCGCTGCCACTGTTTGTCATCACO^

T P O E Y A N S A S L P H V Q V A L R M N R E R N R L

ACTCCCCAGGAATATGCCAATAGTGCTTt^rTGCCCCACGTCCAa?rGGCCCTTCGTATGAATCGCGAGCGTAATAtXXr^

Y K K G D M V D Y V I C L D G T T N L A M Q R A Y H L

TAC^AAAAGGGCGATATGGTGGACTATGT^TCTCTrTTGGATGGGAa^ACAMTT^

D E L K T S E D K K L Q L D T N L L L G H Q I H P V VGATGAGCTCAAGACC^GTGAGGATAAGAAGCTGCAGCTGGACAOiAATCTACTACTGGGACACCAC^TCCATCCGGTAGTC

5646137

732181002991273801544611815422086232357042627852898663169473431028370

1109397

11904241271451

1352478

1433505

1514532

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1757613

1838640

19196672000694

2081721

2162"748

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2567883

2648910

2729937

2810964

28919912972101830531045313410723215109932961126337711533458118035391207362012343701

Fig. 2. Amino acid andnucleotide sequences of thegene for 180 kDa subunit ofthe a polymerase, includingthe 5' untranslated region andalso some of the 5'untranscribed region. Thepositions of transcriptioninitiation and thepolyadenylation signal are bothin bold type.

Gene for 180 kDa subunit of DNA polymerase 851

T R M V E V L E G T D A S R I A E C L G M D P T K F R 1 2 6 1

ACGCGAATGGTGGAGGTGCTGGAGGGAACCGATGCGAGTCG<^TCGCCGAGTGTCTGGGCATGGACCCAACCAAGirrCCGT 3 7 8 2

Q N A Q R T Q R E N T E Q S E G E S L L K T T L Q L Y 1 2 8 8

CAGAATGCGCAGAGAACCCAGCGAGAGAACACCGAGCAGTCGGAGGGTGAGTCCCTGCTC AAGACGACTCTGCAGTTGTAT 3 8 6 3

R L C E P F R F Q C V T C K T E Q L M A S A Y R P G P 1 3 1 5

S N S H I A V L Q Q C A K S D A K R H R F S T W Q R C 1 3 4 2

AGCAACTCCCACATAGCCGTACTCCAGCAATGCGCTAAGTCCGATGCCAAACGGCACCGA'ITCAGTACCTGGCAACGGTGC 4 0 2 5

A I S L Q L S M R Q Y V Q R F Y K N W L V C D H P D C 1 3 6 9

GCAATCAGTCTG<^GCTTTCCATGCGGCAGTATGTGCAACGGTTCTATAAAAATTGGCTGGTCTGCGATCACCCGGATTG^ 4 1 0 6

N F N T R T H S L R K K S H R P L C Q K C R S G T L L 1 3 9 6

AACTTCAACACCCGCACACACTCCTrGAGGAAGAAATCCCACCGCCCGCTGTGCCAAAAGTGCAGGAGCGGCACCCTGCTG 4 1 8 7

R Q Y T E R D L Y N Q L C Y L R F M F D L G K Q T L Q 1 4 2 3

CGTCAGTATACGGAGCGGGACCTGTACAATCAGCTGTOrTACCTGCGATTCATGTTCGACCTC 42 68

Q K P T L T P E L E Q A Y Q L L Y E T V D Q Q L Q S S 1 4 5 0

CAAAAACCCACCCTCACACCGGAGCTGGAGCAGGCCTATCAACTCCTATACGAAACGGTGGATCAGCAGTTGCAGAGCTCC 434 9

S Y V I I S L S K L F A R S A Q M S L Q P S V A Q P Q 1 4 7 7

TCCTACGTGATCATCTCGCTAAGCAAGCK^ITTGCCCGATCCCTGGCTCAGATGTCCCTGCAACCGTCCGTGGCGCAGCCC 44 3 0

L I E A I P S A L A D V V 1 4 9 0

CAGATCGAAGCAATTCCGAGTGCTTTGGCG<^TGTGGTCTAAGTGTTrAATTAACTAGCCrITTGrPTATAAGTTCCATTAAA 4 5 1 1

AATACTACAAAAGAGAAAAAAAAAAAAAAAAAA

embryo (particularly the sites of cleavage) can beobserved to stain more intensely than others.

Simultaneous immunofluorescence and staining ofnuclei with Hoechst suggested that the staining of thenuclei is not constant at all stages of the cell cycle. Sphase and prophase nuclei all appeared to show nuclearstaining (on average this accounted for approximately75% of stage 6-11 embryos). For nuclei in other mitoticstages (approx. 20%) no immunostaining of the apolymerase was observed. This was further confirmedby looking at the small percentage of cycle 6-11embryos showing differential staining (approx. 5%).Those regions of the embryo that showed no nuclearstaining appeared to be regions in the later mitoticstages: metaphase, anaphase and telophase (Fig. 8).

Preliminary studies using higher magnificationssuggest that there is a significant difference in the natureof the staining of nuclei in younger as opposed to olderembryos. For younger embryos, where the rate ofreplication is very fast, staining is uniform throughoutthe nucleus. However, in older embryos the polymerasecan be seen to stain in discrete spots within the nucleus(data not shown).

Discussion

All replicative eukaryotic polymerases so far isolated(a, 5 and e) contain six regions of strong similarity. Theexact significance of these sites is not yet clear;however, their high degree of conservation suggests aninvolvement in the catalytic function directly, or ininteractions with other subunits. The gene that we haveisolated contains these six regions. In addition, it showsstrong similarity outside these regions to the largesubunit of the a polymerases from both yeast (S.cerevisiae and 5. pombe) and man. On this basis weconclude that we have isolated the gene for the largesubunit of the a polymerase of Drosophila melanogas-ter. Past work (Cotterill et al. 1987c) has suggested thatthe DNA replicative machinery of Drosophila showssome differences from other organisms. It has proveddifficult to isolate <5 and e polymerase-like activities; inaddition, unlike the yeast and human arpolymerases theDrosophila a polymerase appears to possess a crypticexonuclease activity associated with the 180 kDa

subunit (Cotterill et al. 1987c). We have thereforeanalysed the sequence in detail to see if any informationcan be gleaned on either of these two subjects. As thispolymerase does not appear to be more 6- or e-like thanother a polymerases so far identified, it seems unlikelythat it possesses a dual role. In addition, althoughsequences are found that are characteristic of exonu-cleases, the position of these sequences in the proteindoes not correlate with their positions in otherpolymerases where a functional exonuclease has beendemonstrated. The functionality of these sites in theDrosophila enzyme therefore remains unclear and afinal solution to this problem will have to await a studyof the properties of the enzyme overexpressed from thecDNA.

Large amounts of the mRNA for the 180 kDa subunitare made in the nurse cells of the ovaries andtransported into the egg. Expression levels then remainrelatively high in embryos when there is a very high rateof replication. One marked exception to this is the 4-8 hperiod when level of mRNA falls. We do not believethis to be caused by a gel artefact, and in fact a similarpattern can be seen in the expression of the mRNA forPCNA (proliferating cell nuclear antigen) in Dros-ophila (Yamaguchi et al. 1990). This probably reflects aswitch between the maternal and the zygotic transcript.The expression of the 180 kDa protein is slightlydifferent. Protein levels rise slightly throughout the firstfew hours of development, presumably to cope with thelarger amounts of template that need to be replicated.This is most likely to be new translation, although wecannot rule out the possibility that in earlier embryos thepolymerase is modified in some way that inactivates it andmakes it inaccessible to the antibody. There is a secondburst of protein in first instar larvae. This is again a stagewhere substantial polytenisation of many larval tissuesoccurs. Since we do not see an equivalent increase in thelevel of mRNA, it is likely that some sort of translationalcontrol operates at this point. These levels of 180 kDaprotein appear to be fairly well parallelled by thepolymerase activity. Although we assayed all aphidicolin-sensitive activity, which could theoretically include a, 6and e polymerases, experiments suggest (Hirose et al.1991) that the a polymerase probably contributes themajor proportion of the activity.

These comparative studies of protein, mRNA and

852 5. Melov and others

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Fig. 3. Comparison of the amino acid sequence of theDrosophila 180 kDa subunit genes with those from yeast(A) and man. (B) The analysis was performed usingMacMolly on the PAM setting with a window size of 20and allowing 7 mismatches. The numbers I-VI refer to thesix defined regions of polymerase homology.

activity levels suggest that the control of the polymerasemay occur in several different ways. Clearly there is acertain amount of transcriptional control, for instancein the ovaries and perhaps at the switch from maternalto zygotic transcription. However, until a functionalassay is performed we can only speculate as to thenature of the cw-acting transcriptional controls in the

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Fig. 4. RNA blot showing developmental alterations in themessage for the 180 kDa subunit. Total RNA was probedwith the full-length cDNA clone as described. The first 6lanes show the embryonic stages, the time in hours isshown above each lane. 1st, 2nd and 3rd represent thethree larval instar stages, ep, early pupal; lp, late pupal;m, adult males; and f, adult females. The position of theRNA Mr markers are shown at the left-hand side of the gel(in kb). Comparison of the size of the message for the 180kDa subunit with that of RNA markers suggested that thegene codes for an mRNA of approximately 4.8 kb. Thesize of the message was confirmed by analysing the sameblot using probes for Drosophila topoisomerase II andcopia (transcript sizes 5.1 kb and 5.0 kb, respectively).Both of these gave bands slightly larger than that for the180 kDa subunit (data not shown).

upstream region of the gene. One possible regulatoryelement is an A+T-rich region that is conservedbetween this gene, the 73 kDa subunit and the PCNAgene. For the PCNA gene it has been suggested thathomeoproteins act through binding to this region,although the mechanism of this still remains unclear.There are also several additional sequences in theupstream region of the gene that are also seen upsteamfrom other Drosophila replication genes (e.g. PCNA,topoisomerase II, 73 kDa subunit). Some of theseregions are short (8-10 bp). However, the cell-cyclecontrol signal for yeast replication genes (the Mlu box)is only 6 bp in length (White et al. 1991). Thesignificance of these short repeats should becomeclearer as the genes for more Drosophila replicationproteins are analysed.

The accumulation of protein in the larvae and pupaemay suggest some form of translational or post-

Gene for 180 kDa subunit of DNA polymerase 853

200

100

a- E -

Fig. 5. Graph showing the comparative expression for themRNA (•) , protein ( • ) , and the aphidicolin-sensitivepolymerase activity (O). The relative data for the RNAand protein lanes were obtained by densitometric scanningof gels. The polymerase activity was measured by timecourse as described in Materials and methods, and eachpoint corresponds to the average of at least 3 sets ofmeasurements. The first 6 sets show the embryonic stages,the time in hours is written below each set. 1st, 2nd and3rd represent the three larval instar stages, ep, early pupal;lp, late pupal; m, adult males; f, adult females. In eachcase the values are expressed as a percentage of the valuefor the 0-4 h embryonic stage.

translational control. The nature of the translationalcontrol is not known. Post-translational controls havebeen seen in other organisms, e.g. phosphorylation(Cripps Wolfman et al. 1989) or glycosylation (Hsi et al.1990) for the human enzyme. Regions of the proteinthat would be susceptible to modifications by suchmechanisms can be seen in the amino acid sequence.However, whether such changes occur for Drosophila

Fig. 6. Distribution of 180 kDa mRNA and protein in ovaries. (A) Stages 1-6; (B) stage 10; (C) late stage 12;(D) localisation of protein. Stages of oogenesis are labelled. Both RNA and protein hybridisations were carried out asdescribed in Materials and methods. For RNA localisation the probe corresponded to the 800 bp piece between the Xholand WiVidlll sites (see Fig. 1). Protein localisations were performed with a monoclonal antibody. P, pole cell nucleus; O,oocyte.

854 S. Melov and others

Fig. 7. Distribution of 180 kDa protein in various embryonic stages; (A) precycle 6; (B) cycle 8; (C) cycle 10; (D) cycle 12;(E) cycle 14. The analysis was carried out as described in Materials and methods, using the same monoclonal antibodyused in Fig. 6.

Fig. 8. Differential staining of embryos with monoclonal antibodies against the 180 kDa subunit of the a- polymerase asrelated to various stages of the cell cycle. (A and C) Hoechst staining of the embryos. (B and D) Immunofluorescencestaining of the same embryos. (A/B) Positively stained S phase/interphase nuclei at the anterior pole with non-stainingmetaphase nuclei in the rest of the embryo. (Qt>) Positively staining prophase nuclei at the anterior pole of the embryowith a progression through anaphase, telophase and metaphase (all non-staining) towards the posterior pole.

Gene for 180 kDa subunit of DNA polymerase 855

in response either to development or to different phasesof the cell cycle has yet to be determined.

The 180 kDa subunit appears to be predominantly anuclear protein, and in fact there are several regions onthe protein that could act as nuclear localisation signals.Staining appears weaker during those mitotic phases ofthe cell cycle when the nuclear membrane has degener-ated. Whether this is active movement or just passivediffusion away from the concentrated sites of its activityis not clear. It is also possible that the diminution ofstaining could be due to modification of the enzymeaffecting the sites to which antibodies bind, althoughthe observation of the same phenomenon with morethan one antibody makes this explanation less likely.Differences in the staining pattern of nuclei can also beseen between different embryos up to cycle 10 and atthe syncytial blastoderm. In older nuclei when the rateof replication is lower the polymerase can be observedto be concentrated at points in the nucleus, whereas inyounger embryos the staining appears to be homo-geneous. This could be just an artefact of the stainingtechnique used. Alternatively, the concentrated pointsof staining could represent polymerase accumulated atclusters of origins on the nuclear matrix. Similarobservations have been made in other organisms (Foxet al. 1991). These sites may not be visible in youngerembryos, since additional origins of replication presentat early stages may necessitate additional 'matrixattachments points'. The increased packing of thesesites may make the boundaries of them indistinguish-able from each other at the levels of magnification used.

During the preparation of this manuscript, Hirose etal. (1991) also presented data on the cloning andanalysis of the 180 kDa subunit of the Drosophila apolymerase. The protein coding sequence that theypresent is substantially the same as ours (96%), and hasthe same postulated transcription and translationinitiation sites. However, we do see some significantdifferences at the 3' end of the gene. At amino acid 1393our sequence deviates from theirs, and readjusts againas position 1426. In addition, our sequence betweenthese sites is 20 amino acids shorter. In the interveningregion the two amino acid sequences show no similarityto each other, although the sequence that we present inthis paper does show homology to both the yeast andthe human proteins. It is possible to get some of thesequence that they present by switching into a differentreading frame, but we cannot explain the discrepancy inthe numbers of amino acids. There also appear to bedifferences in the restriction pattern of the genomicDNA at the 3' end of the gene. In addition there aresignificant differences in the size of the mRNA (4.8 kb(this paper) versus 5.7 kb (Hirose et al. 1991)), and themRNA developmental profiles. Further analysis of thegene for the 180 kDa subunit may help to explain someof these discrepancies.

We would like to thank Tamsin Majorus for help with the insitu's, and David Glover, David Hartley and Kevin O'Harefor helpful discussions. We would also like to thank NickBrown and Jorg Hoheisel for providing libraries. SM and HV

were supported by grants from the CRC and the MRCrespectively.

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(Received 4 February 1992 - Accepted 13 April 1992)