Stage specific expression of proliferating cell nuclear antigen and DNA polymerase δ from Plasmodium falciparum

:

Molecular and Biochemical Parasitology 79 (1996) 177- 182

Stage specific expression of proliferating cell nuclear antigen and DNA polymerase 6 from Plasmodium falciparum Paul Horrocks”, Institute

Mandy

Jackson’,

of Cell and Molecular

Biology.

Sandie University

Cheesman, of Edinburgh,

John H. White, Mavfieid

Road Edinburgh

Brian J. Kilbey EH9 3JR UK

Received 21 March 1996; revised 8 May 1996; accepted 9 May 1996

Abstract Antisera raised against proliferating cell nuclear antigen (PfPCNA) and DNA polymerase S (PfDNA Po16) have been used against extracts from synchronised parasites to show that both proteins accumulate in trophozoites and persist in schizonts. The steady-state transcripts from both PfPCNA and PfDNA Po16 also accumulate at the trophozoite stage. However, nuclear run on analysis shows that, whereas PfDNA Po16 promoter activity is absent in rings but present in trophozoites and schizonts. the PfPCNA promoter is active throughout the intraerythrocytic cycle. This suggests that mechanisms regulating the expression of these two genes may be different although their coordinated activity is required for DNA replication. DNA

Keywords:

polymerase

6; Plasmodium falciparum;

Proliferating

Evidence

from

the

chromosomal

SV40 DNA

model

system

replication

for eusuggests

Abbreviations: ORF, open reading frame; PCR, polymerase chain reaction; sdH,O. sterile distilled water: UTR, untranslated region. * Corresponding author. Tel.: +44 131 6505395; fax: +44 13I 6683870; e-mail: [email protected] ’ Present address; Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK 0166-6851/96/$15.00 PI1 S166-685

0 1996 Elsevier Science B.V. All rights

1(96)02657-6

antigen;

Stage-specific

expression

the DNA polymerase cc/primase complex is essential for the initiation of eukaryotic DNA replication it is rapidly replaced by the more processive DNA Polymerase 6 (DNA Po16). It is this enzyme which is probably responsible for the major part of leading and lagging strand synthesis [ 1,2]. Proliferating cell nuclear antigen (PCNA) plays an essential role in the DNA polymerase 6 activity by loading the polymerase at the primer terminus and increasing its processivity. A number of genes encoding replication proteins of P. falciparum have now been isolated that

1. Introduction

karyotic

cell nuclear

reserved

although

178

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et al. / Molecular

and Biochemical

and characterised, including the two major DNA polymerases and PCNA [3-61, but little is yet known about their expression during the complex life cycle of this parasite. Most work has been done on the intraerythrocytic stages of the parasite’s development where it has been shown that DNA synthesis starts in the trophozoites and continues in schizonts. Jaikaria et al. [7] have shown that the transcript for DNA Polymerase CI is absent from ring stage parasites and then accumulates in trophozoites and schizonts but there is no evidence concerning the levels of the enzyme itself. Indirect immunofluorescence assays with antisera against PfPCNA show that the protein starts to accumulate in the mid-trophozoite stage but there is no data available regarding stage specific transcript accumulation [6]. PfDNA Po16 expression has not been studied. Here we provide a more complete picture of the regulation of PfPCNA and PfDNA PolS during the intraerythrocytic cycle, which, by analogy, are considered to co-operate during DNA synthesis.

2. Materials and methods 2.1. Purasite

culture

The Kl isolate [8] of P. fdciparum was used throughout. Parasites were cultured and synchronised using the methods of Zolg et al. [9] and Lambros and Vandenberg [lo]. 2.2. Northern

blot analysis

Total RNA was isolated from synchronised parasites by the acidified guanidine isothiocyanate-phenol-chloroform method [l 11. Ten micrograms of total RNA from each major stage (rings, trophozoites and schizonts) were size fractionated on a 1% agarose gel containing 0.7% formaldehyde in MOPS buffer (20 mM MOPS, pH 7.0, 5 mM sodium acetate, 1 mM EDTA) and transferred to a Genescreen Plus membrane (DuPont) by capillarity in 10 x SSC (1 x SSC is 150 mM NaCl, 15 mM trisodium citrate, pH 7.0). Membranes were pre-hybridised overnight at 60°C in 20 mM sodium phosphate (pH 7.2), 7%

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79 (1996) 177-l 82

SDS. The probes were added to the same solution and incubated for 16 h at 60°C. The probes used were either purified products of a polymerase chain reaction (PCR) or cloned DNA fragments radiolabelled with 32P by randomly primed DNA synthesis [12]. Membranes were washed at 60°C for 20 min, twice, in each of 2 x SSC, 0.1% SDS and 0.5 x SSC, 0.1% SDS and once in 0.1 x SSC, 0.1% SDS. Film was exposed to the washed membranes at - 70°C with an intensifying screen. Before reprobing, membranes were stripped by incubation at 70°C for 1 h in 10 mM Tris-HCl (pH S.O), 1 mM EDTA, 1% SDS, and checked by overnight exposure at - 70°C. 2.3. Nuclear run on analysis All steps were carried out on ice. 5 x 10’ parasites at each major developmental stage were harvested and washed once in PBS buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM disodium hydrogen orthophosphate, 1.4 mM potassium dihydrogen orthophosphate). Erythrocytes were lysed by the addition of 5 ~01s. of 1 x SSC, 0.1% saponin. The released parasites were washed once in buffer A (20 mM PIPES pH 7.5, 15 mM NaCl, 60 mM KCl, 14 mM 2-mercaptoethanol, 0.5 mM EGTA, 4 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 0.125 mM phenylmethylsulphonyl fluoride). The washed parasites were resuspended in 3 ml of buffer A and transferred to a dounce homogeniser. Two-hundred microlitres of a 10% NP-40 solution were added and 20 strokes with a B pestle applied to break open the parasites. The nuclei were collected (12000 x g for 10 min at 4°C) and washed once with buffer A. The nuclei were allowed to transcribe for 10 min at 37°C in 600 ~1 of buffer B (50 mM HEPES pH 7.9, 50 mM NaCl, 10 mM MgCl,, 1.2 mM dithiothreitol, 10 mM creatinine phosphate, 1 mM GTP, 1 mM CTP, 4 mM ATP, 25% glycerol, 25 U ml ~ ’ RNasin (Promega), 0.2 mgml - ’ creatinine kinase, 0.5 PM [x-~~~]UTP 3000 Ci mmol-‘). Radiolabelled RNA was isolated as described [l 11. The nascent RNA was hybridised to a membrane, to which 1 pg of single stranded DNA from the genes of interest had been fixed, as described in Section 2.2. The filter was hybridised

P. Horrocks et ui.

I Molecular and Biochemical Parasitology 79 (1996) 177- 182

for 24 h at 60°C. The filter was washed and exposed to film as previously described.

3. Results

2.4. DNA polymeruse 6 f&ion und antibody production

3.1. DNA polymerme speciJic

protein

constructs

A 599 bp PCR product (nt 1146-1745 of PfDNA Pol6 ORF) amplified from the cloned gene was sub-cloned directly into the pCRScriptTM SK + vector (Stratagene). A 618bp BamHI-SmuI fragment containing the PCR product in frame was sub-cloned into the pGEX1 vector, to give the construct pGEX1 /S6, and transformed into Escherichiu coli [BL21(DE3) pLysS]. Individual transformants were induced with 0.1 mM isopropyl-1-thio-B-D-galactoside for 3 h. An insoluble product of the expected size of 58.5 kDa was identified by SDS PAGE analysis. Approximately 100 pg of gel purified fusion protein from inclusion bodies was emulsified in a mixture of 0.5 ml of PBS and 0.5 ml Freund’s complete adjuvent and used to immunise a New Zealand White rabbit. Three booster immunisations were given of approximately 60 pg of fusion protein in Freund’s incomplete adjuvent at 3-week intervals. Blood samples were removed between booster immunisations to monitor the immune response by western blotting. Antiserum taken after the third boost was used for subsequent analysis. 2.5. Western analysis A cell extract was prepared from 2 x lo9 parasites from each of the major developmental stages. Erythrocytes were lysed by the addition of 5 ~01s. of 1 x SSC, 0.1% saponin and washed once in PBS. Parasites were lysed in 200 ~1 of 1 x ioading buffer (62.5 mM Tris-HCI (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol). Genomic DNA was sheared by repeated passages through a 25-gauge needle. Equal volumes of the parasite extract from each stage were fractionated by SDS PAGE on an 8% gel in running buffer (25 mM Tris, 192 mM glycine, 1% SDS). The proteins were transferred to Hybond-C (Amersham), blocked and probed with a 1:1000 dilution of anti-PfPCNA and anti-PfDNA Po16 serum as described by Kilbey et al. [6].

6 expression

179

is stage-

A 31 amino acid region from the murine DNA polymerase 6 sequence has been shown to be highly immunogenic [13]. The nucleotides encoding this region in the PfDNA PolS sequence were identified, amplified by PCR and expressed as a GST fusion protein (pGEX1/66). The antiserum obtained following immunisation with this fusion protein detected the 120 kDa full length recombinant PfDNA Po16 (data not shown). When a blot of total parasite extract from an asynchronous culture was used with anti-PfDNA PolS the expected band of 120 kDa was also detected (data not shown). The pre-immune serum failed to detect a band of this size from either western blot. Total protein was isolated from the same numbers of parasites in each of the principle stages of the intraerythrocytic cycle, size fractionated and western blotted before probing with anti-PfDNA Po16 and anti-PfPCNA serum [6] (Fig. 1). The results of the anti-PfPCNA serum agree with the data previously obtained by indirect immunofluorescence assays. A band of the predicted size, of approximately 30.5 kDa, can be just detected in rings but protein levels increase dramatically in trophozoites and schizonts. The 120 kDa STRM 4-175 4-120 +a3

32.5 * -30.5 +25 Fig. 1. Western analysis of P. falciparum synchronised intraerythrocytic stage culture. (A) Anti-PfDNA Po16, and (B) Anti-PfPCNA Pold. The percentages of each stage (ring:trophozoite:schizont) sampled were: R, rings (88:12:0); T, trophozoites (5:87:8); S, schizonts (3:26:71). M. prestained markers (Biolabs) indicted in kilodaltons

180

P. Horrocks et al. 1 Molecular and Biochemical Parasitology 79 (1996) 177-182

R

T

S 42.2

A. PCNA

41.85

B.ActinI

42.5

* Z-3.8

C. 3.0 gene

D. GBP130

T

R E.PCNA

s 42.2 41.85

,

I

F. DNA Po16 * * ‘. L

45.2

Fig. 2. Northern analysis of P. falciparum synchronised intrderythrocytic stage culture. Sizes are indicated in kilobases. The percentage of each stage (ring:trophozoite:schizont), for (A-D), sampled were: R, rings (79:20:1): T, trophozoites (5:81:14); S, schizonts (4:12:84). For (E,F): R, rings (87:13:0); T, trophozoites (6:85:9): S, schizonts (0:28:72).

band detected by the anti-PfDNA Po16 serum follows a similar pattern as for the anti-PfPCNA serum. The faint signals observed with both antisera to ring stage extracts are probably the result of trophozoite contamination of this stage or perhaps a low level of expression of both these proteins. 3.2. DNA polymerase 6 and PCNA accumulate in tropliozoites

transcripts

A northern blot analysis of total RNA from parasites from each of the major mental stages of the intraerythrocytic shown in Fig. 2. The PfPCNA transcripts,

isolated developcycle is sized at

1.85 kb and 2.2 kb, are apparently present in all stages but accumulate significantly in the trophozoite stage. Microscopic examination of the samples reveal a contamination of both the ring and schizont stages by trophozoites, this is the result of the difficulty in achieving complete synchrony with large volumes of culture. A series of probes, specific to different stages of the intraerythrocytic cycle, was used against the membrane to indicate whether the signals obtained from the PfPCNA probe in the ring and schizont samples were due to trophozoite contamination. The Actin I transcript, 2.5 kb, accumulates only in the trophozoite and schizont samples [14]. The 3.8 kb gene transcript, accumulates in rings and trophozoites [15]. The GBP130 gene probe a 6.6 kb transcript, which only accumulates in trophozoites [I 51, is seen to follow the same pattern of accumulation as PfFCNA. Using a second northern blot where the trophozoite contamination of the ring sample is substantially reduced the PfPCNA signal is also proportionately reduced. These data would suggest that the PfPCNA transcript only accumulates in the trophozoite stage, although low levels of transcript accumulation in rings and trophozoites may be present. The PfDNA Pold transcript of 5.2 kb only accumulates in the trophozoite stage. 3.3. Nuclear run on analysis Promoter activity of the genes encoding PfPCNA and PfDNA Po16 was analysed using nuclear run on experiments. Nuclei were isolated from synchronised parasites in the ring or a mixture of trophozoite and schizont stages and in vivo radiolabelled transcripts prepared from the two samples. These were hybridised to single stranded immobilised PCR products, each of approximately 1 kb, and each derived from one of the coding sequences being studied (Fig. 3). No signals were detected from the PfDNA Polb promoter in ring stage nuclei, there was, however, promoter activity in the trophozoite/schizont nuclei preparation. In contrast, PfPCNA promoter activity was detected in both the ring and trophozoite/ schizont nuclei preparations. Although equal amounts of nuclei from each of the two

P. Horrocks

et al. / Molecular

and Biochemical

synchronised stages were used in the hybridisations, the experiment was repeated with MSP-1 (P195) as an internal control. The MSP-1 promoter is reported to be equally active in all intraerythrocytic stages [ 16,171. In both experiments a sample of genomic DNA was used to demonstrate that overall transcriptional activity in trophozoites and schizonts is greater than that of the ring stages. pBluescriptIISK + (Stratagene) was used as a negative control.

4. Discussion We have shown that PfPCNA and PfDNA PolS protein levels increase from very low levels in rings to high levels in trophozoites and schizonts. The transcript levels for both PfPCNA and PfDNA Po16 show a similar increase as the parasite develops into a trophozoite, but these

A

R

T/S

PCNA DNA Po16 gDNA pBSIISK+

B

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79 (1996) 177-182

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levels substantially decrease in the schizonts. In contrast to this, promoter activity for these two genes behave differently. There is no detectable PfDNA Pol6 promoter activity in the ring sample but a dramatic increase is observed in the trophozoites/ schizont sample. The promoter for PfPCNA, in contrast, remains active in both the ring and trophozoite/schizont samples. This suggests that although transcript and protein levels increase in parallel in preparation for DNA synthesis the mechanism by which this is brought about is different for PfPCNA and PfDNA Po16. The parallel increase in transcript level and promoter activity for PfDNA Po16 suggests that message levels are regulated at the level of transcription initiation, whereas PfPCNA transcript levels must be regulated post-transcriptionally. How the transcript is post-transcriptionally regulated will be examined further. The PCNA promoter in humans and mice is active in the G, phase of the cell cycle although no transcripts accumulate. Only when the cells are stimulated by serum or growth factors to enter the cell cycle do transcripts accumulate at the GljS phase boundary [18,19]. One interesting observation from these studies is that the stage specific western data indicates that both the PfPCNA and PfDNA Po16 protein are found at approximately the same level in trophozoites and schizonts even though the transcripts are probably only present in trophozoites. Presumably sufficient levels of polypeptide must be translated, while the transcripts are present in trophozoites, to complete schizogony.

PCNA Acknowledgements P195 gDNA pBSIISK+

Fig. 3. Nuclear run on analysis of P. jhlciparum synchronised intraerythrocytic stage culture. (A) Initial comparison of PfPCNA and Pf DNA Po16. (B) Confirmation of PfPCNA with P195. The percentage of each stage (ring:trophozoite:schizont) sampled were: R, rings (91:9:0); T/S, trophozoites/schizonts (2:62:34). pBSI1 SK + . pBluescriptI1 SK +

Michael Lanzer generously supplied the Actin I, P195, GBP130 and 3.8 gene probes, protocols and advice. We would like to thank Kerrie Tosh for assistance and critical reading of the manuscript, Ann-Marie Ketchen with P. fulcipurunz culture and Jennifer Daub for supplying the full length recombinant PfDNA PolS. Research in this laboratory is supported by the Medical Research Council. PH was supported by a MRC studentship.

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