in situ detection of pbs21 mrna during sexual development of plasmodium berghei

ELSEVIER Molecular and Biochemical Parasitology 68 (1994) 189-196

MOLECULAR AND

BIOCHEMICAL PARASITOLOGY

In situ detection of Pbs21 mRNA during sexual development of Plasmodium berghei

Joanne Thompson *, Robert E. Sinden Department of Biology, Imperial College, Prince Consort Road, London, SW7 2BB, UK

Received 31 May 1994; accepted 5 August 1994

Abstract

The patterns of expression of ribosomal RNA and of mRNA encoding the parasite surface antigen Pbs21 have been investigated during the sexual stages of development of the malaria parasite, Plasmodium berghei, using the technique of non-radioactive in silu RNA hybridisation. An RNA probe complementary to a region of the small subunit of P. berghei ribosomal RNA hybridised to parasites at all stages of development in a smear of blood taken from mice infected with P. berghei. Messenger RNA encoding Pbs21, in contrast, was detected only within parasites committed to sexual development within the vertebrate host and, furthermore, was shown to be expressed in a sex-specific manner, exclusively within female gametocytes. At later stages of sexual development, Pbs21 mRNA was detected at high levels in female gametes and ookinetes. We have previously shown that Pbs21 protein is first detectable only after the initiation of gametogenesis which occurs following transmission to the insect vector. These results suggest, therefore, that post-transcriptional mechanisms operate to regulate the translation of Pbs21 mRNA as it accumulates during female gametocytogenesis.

Keywords: Plasmodium berghei; Pbs21; In situ hybridisation

1. Introduct ion

Malaria parasites proliferate asexually within the hepatocytes and then in the red blood cells of infected vertebrate hosts, but must produce gametocytes in a sexual cycle of development for transmission through the mosquito vector. The differentiation of gametocytes into gametes within the vector is regulated by extraneous factors which ensure that mature gametocytes are triggered to emerge from the

* Corresponding author. Tel.: 071 594 5423; Fax: 071 5945424; E-mail: [email protected].

vertebrate red blood cells and undergo gamete for- mation only when they encounter conditions similar to those found in the mosquito midgut [1-3]. After ingestion within the mosquito blood meal, gametes are immediately vulnerable both to degradation by the proteolytic enzymes of the midgut [4] and to recognition and destruction by various components of the vertebrate host immune system which are also taken up in the blood meal. Antigens expressed in the gametocyte are capable of invoking an immune response in the vertebrate host. If such antigens are expressed on the extracellular malarial gametes in the mosquito blood meal, antibodies to these antigens could suppress transmission [5]. It has been

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190 J. Thompson, R.E. Sinden / Molecular and Biochemical Parasitology 68 (1994) 189-196

suggested, therefore, that the survival of the parasite in the blood meal is dependent upon the rapid expression of novel mosquito stage-specific parasite- surface antigens only after transmission to the mosquito vector has taken place [6].

Transmission of the parasite through the mosquito vector can be blocked by the presence, within the infectious blood meal, of antibodies which recognise antigens expressed during the gametocyte and gamete or the gamete/ookinete stages of the parasite's development [6]. Amongst the latter group of antigens, experiments carried out using the major zygote and ookinete cell surface protein of Plasmodium falciparum, Pfs25, have provided evidence of the potential efficacy of transmission blocking vaccines [7-9]. Pfs25 protein is not detectable on the surface of gametocytes but appears within minutes of the activation of gametogenesis and is then synthesised at a constant and high rate [7,10]. A second, closely related P. falciparum ookinete-surface protein, Pfs28, has recently been identified (D.C. Kaslow; personal communication). Its homologue, Pbs21, in the rodent malaria species P. berghei [11,12], is capable of eliciting a similarly strong transmission- blocking response within immunised mice [12-14].

Pbs21 appears on the surface of female P. berghei gametes, following their emergence from the red blood cell, but cannot be detected within or on the surface of gametocytes or male gametes [11,12]. Northern blot analyses have, in contrast, shown that a 1.5-kb mRNA transcript encoding Pbs21 accumulates within P. berghei parasites whilst they are still within the infected mouse host [15]. Together, therefore, these observations have provided an indication that the expression of sexual-stage gene products may be subject to regulation by post-transcriptional mechanisms. It is not possible to formally show using Northern blot, however, which populations of parasites are expressing Pbs21 mRNA nor to exclude the possibility that the mRNA detected has been synthesised within spontaneously activated gametocytes and gametes. In addition, these analyses do not show whether the female-specific expression of Pbs21 protein results directly from transcription of the Pbs21 gene only within female gametocytes and gametes or, alternatively, occurs because Pbs21 mRNA is expressed in both male and female gametocytes but is translated only in female gametes.

As a first stage in investigating the regulation of gene expression in the parasite, we have directly determined the pattern of expression of Pbs21 mRNA at the individual cellular and sub-cellular level using the technique of in situ hybridisation (see reference [17] for an extensive review of the in situ hybridisation procedure). In situ hybridisation studies give data on the cell-type, stage-specificity and cellular localisation of mRNA expression which cannot be obtained by Northern blot analyses relying on the use of mixed parasite populations. Furthermore, the relative levels of expression of different RNAs within individual cells can be determined by concurrent hybridisation of two probes. We have used this procedure, therefore, to discriminate precisely at which stages of their development P. berghei parasites express mRNA transcripts encoding Pbs21. We show that Pbs21 mRNA is expressed exclusively within macrogametocytes during the vertebrate stages of the parasite's life cycle and, following gametocyte activation and fertilisation, is expressed at high levels during the development of ookinetes.

2. Materials and methods

2.1. Parasites

TO mice were infected with P. berghei ANKA clones 2.34 or 2.33 (high and non-gametocyte pro- ducers respectively) to obtain blood smears of asexual parasites and gametocytes. Gametes and ookinetes of clone 2.34 were prepared by incubating blood samples containing mature gametocytes in ookinete culture medium for 10 min (gametes), or 19 and 24 h (immature and mature ookinetes) at 19°C [16].

2.2. Synthesis of RNA probe

A 670-bp fragment from the cDNA clone A2TS [15], containing the coding sequence of mouse Pbs21, subcloned into pBluescript SK, (Stratagene) was lin- earised by cleavage at the internal HindlII or PstI sites. Transcription using T 3 o r T 7 RNA polymerase (Promega) generated a 370-nucleotide antisense and a 480-nucleotide sense probe, respectively. A tem- plate for the transcription of a Plasmodium-specific rRNA probe was prepared by PCR amplification

J. Thompson, R.E. Sinden / Molecular and Biochemical Parasitology 68 (1994) 189-196 191

from a cDNA clone containing an 8.8-kb fragment of the A-type small subunit of the rRNA gene from the P. berghei NYU2 strain (generously provided by A. Waters). During the first round of amplification, a 5' oligonucleotide with the sequence CTTAAC- CATAAACTATGCCG was used in conjunction with a 3' oligonucleotide, GACTCACTATAGGGCG- GTACTGAAGGAAGC, which contains 15 bp of the T 7 RNA polymerase site. The remainder of the T 7

RNA polymerase site and 10 bp of protecting se-

la

g

quence was added during a second round of PCR amplification using the 3' oligonucleotide, ACAAGCTTCTAATACGACTCACTATAGGGC. Transcription using T 7 RNA polymerase generated a 92 nucleotide anti-sense probe. RNA probes were synthesised in vitro in the presence of digoxigenin, biotin or fluorescein UTP (Boehringer) as described by the manufacturers. Unincorporated nucleotides were removed by ethanol precipitation in the presence of 0.5 M LiC1.

b

Fig. 1. Detection of RNA in P. berghei. In situ RNA hybridisation to blood smears taken from a mouse infected with P. berghei ANKA clone 2.34 (1.1 and 1.3) or clone 2.33 (Fig. 1.2). Panels (a) show the signal obtained in gametocytes (g), rings (r), trophozoites (t) and schizonts (s) following hybridisation with a digoxigenin-labelled RNA probe, specific for P. berghei SSu rRNA, detected with a rhodamine-conjugated anti-digoxigenin antibody. A biotin-labelled anti-sense RNA probe specific for Pbs21, detected with a fluorescein- conjugated anti-biotin antibody, hybridises solely to gametocytes (Fig. 1.1b and 1.2b). A biotin-labelled sense RNA probe for Pbs 21, detected in the same way, does not hybridise with gametocytes (Fig. 1.3b). Scale bar: 5 /.tm.


2.3. In situ hybridisation and detection 3. Results

The protocols of Brahic and Ozden [17] and Wilkinson [18] were adapted for use with thin-film smears of Plasmodium-infected blood cells. Blood films were prepared on slides coated with 0.02% polyvinyl pyrrol idone/0.02% Ficol l /0 .02% bovine serum albumin in 3 × SSPE (0.54 M NaC1/0.03 Na H2PO4/ 0.03 M EDTA, pH 7.4). Cells on slides were fixed in 100% ethanol for 5 min at 4°C, hydrated through a series of 90%, 70% and 50% ethanol for 5 min each and then fixed again in 4% paraformaldehyde in phosphate-buffered saline (PBS-A) for 20 min. After fixation, cells were incubated with 5 /xg m1-1 proteinase K in 0.1 M Tr i s / 0.01 M EDTA, pH. 8.0 for 30 min at 37°C; following this treatment the parasites were optimally per- meabilised to allow access of the probe but appeared to retain good morphology within the red blood cells. The cells are then washed for 5 min in 2 X SSPE, transferred to 0.2 M HC1 for 15 min and washed again in 2 X SSPE. Cells were incubated in 0.1 M triethanolamine, pH. 8.0, containing 0.25% acetic anhydride with stirring for 5 rain. The same amount of acetic anhydride was then added with stirring for 5 min and cells were washed finally in 2 X SSPE.

RNA probes were denatured by heating at 65°C for 5 min and added at a final concentration of 1-2 ng /xl 1 to the hybridisation solution (50% formam- i d e / 2 X SSPE/0.1% bovine serum albumin/0.2% polyvinylpyrrolidone/0.2% Fico l l /10% dextran sulphate/0.5% SDS/200 /xg m1-1 yeast tRNA. A 1 cm z area of the blood smear was covered with 50 /xl of hybridisation solution containing probe and a siliconised coverslip, and the slides were incubated in a humidified box at 50°C for 14-16 h. After hybridisation the cells were washed for 1 h each in 2 × SSPE at 23°C, 0.2 × SSPE at 50°C and 0.2 x SSPE at 23°C. The cells were equilibrated in PBS-A containing 0.1% Tween (PBS-AT) for 30 min and then incubated in PBS-AT containing, where appropriate, 10 /xg ml-1 of rhodamine conjugated anti- Digoxigenin Fab fragment (Boehringer) and 10 /xg ml 1 fluorescein conjugated anti-biotin antibody (Vector) for 1 h. The cells were washed in 3 changes of PBS-AT for 20 min each, mounted in Vector- Shield and visualised by Confocal Laser Scanning Microscopy (Bio-Rad).

3.1. In situ detection of RNA in Plasmodium

Messenger and ribosomal RNAs were detected in parasites within a blood smear taken from mice infected with P. berghei by direct hybridisation with a digoxigenin or biotin-labelled single stranded RNA probes which was visualised using appropriate rhodamine or fluorescein-conjugated secondary antibodies. Parasites at all stages of development could reliably be identified by the pattern of signal visible following hybridisation with a hapten or fluo- rochrome-labelled RNA probe which is complementary to a 90-nucleotide region of the mature form of the small subunit of P. berghei ribosomal RNA (SSu rRNA). SSu rRNA is distributed throughout the cytoplasm of all parasites but is present at very low levels in the nucleus and is not present in intracellu- lar vacuoles. Parasites could, therefore, be differenti- ated by their characteristic size and shape, the size of their nucleus and the presence of vacuoles (Fig. 1. panels la, 2a and 3a). All subsequent in situ hybridisation reactions were carried out using this SSu rRNA probe in conjunction with a single-stranded RNA probe specific for Pbs21, labelled with a complementary hapten.

3.2. Pbs21 mRNA is expressed in sexual-stage parasites

To determine which parasites express Pbs21 mRNA in the vertebrate host, a hapten-labelled RNA probe specific for Pbs21 was hybridised to cells in smears of blood taken from mice infected with either the P. berghei A N K A clone 2.34, which readily produces gametocytes, or the non-gametocyte pro- ducing clone, 2.33. An anti-sense Pbs21 RNA probe hybridised specifically to a sub-population of parasites of clone 2.34 (Fig. 1. panel lb) but did not hybridise to any of the parasites of clone 2.33 (Fig. 1. panel 2b). A sense RNA probe for Pbs21, used as a control, did not hybridise with any cells of clone 2.34 (Fig. 1. panel 3b). Parasites showing a positive signal after hybridisation with an anti-sense Pbs21- specific RNA probe were large, unvacuolated, pigmented cells with a small nucleus showing all the morphological characteristics of mature, unactivated


gametocytes (Fig. 1. panel la). It is clear, therefore, that the only cells expressing Pbs21 m R N A during the vertebrate blood-cell stages of the parasite 's development are gametocytes.

Pbs21 m R N A is detected throughout the cytoplasm of gametocytes in a pattern of discrete dots but is present at almost undetectable levels within the nucleus. Since it is not possible, at this level of resolution, to determine whether the subcellular morphology of the parasites has been maintained throughout the hybridisation procedure, the pattern of Pbs21 m R N A distribution within the cytoplasm cannot be reliably interpreted. The inability to detect significant levels of Pbs21 m R N A within nuclei, however, is unlikely to be an artifact due to poor penetration of the probes through the nuclear mem- brane as we have shown that it is possible to detect the expression of unspliced precursors of ribosomal RNA transcripts within the nucleus using a similar sized probe (290 nucleotides) with the same procedure (unpublished results). At this level of resolution, therefore, there is no evidence to suggest that

Pbs21 m R N A is retained within the nucleus of gametocytes in an unprocessed form or that it is stored within major compartments within the cytoplasm.

3.3. Pbs21 mRNA is expressed only in female gametocytes

Female and male gametocytes can be differenti- ated both by their morphology under phase contrast microscopy and by their appearance after hybridisation with an SSu rRNA-specific fluorescently labelled probe. Mature male and female gametocytes are both large pigmented forms which fill the red blood cell. Male gametocytes, however, have rela- tively large nuclei compared to those of females. In addition and in agreement with previous reports [19], mature male gametocytes contain far fewer ribosomes than female gametocytes and asexual parasites. Male and female gametocytes were, therefore, easily distinguished by their nuclear size and by the relative intensities of the signal obtained following hybridisation with a SSu rRNA-specific probe (Fig.

b

Fig. 2. Sex-limited expression of Pbs21 mRNA. In situ hybridisation to female (Fig. 2.1) and male (Fig. 2.2) gametocytes of P. berghei clone 2.34. All parasites hybridise with a digoxigenin-labelled SSu rRNA probe, detected with a rhodamine-conjugated anti-digoxigenin antibody (panels la and 2a). Female and male gametocytes (fg and mg), rings (r) and a trophozoite (t) are depicted. A Pbs21-specific biotin-labelled probe, detected with a fluorescein-conjugated anti-biotin antibody hybridises exclusively with female gametocytes (Fig. 2.1b). Scale bar; 5 /xm.


2. panel la and 2a). Expression of Pbs21 mRNA could be detected only in gametocytes which were identified as female (Fig. 2. panel lb) and could not be detected in a population of gametocytes which were identified as male (Fig. 2. panel 2b) by all the above criteria. These results clearly show, therefore, that the expression of Pbs21 mRNA is controlled in a stage and sex-limited manner.

3.4. Expression of Pbs21 mRNA in mosquito-stage

parasites

The pattern of expression of Pbs21 mRNA in mosquito-stage parasites was investigated by in situ

2

N

RNA hybridisation to cells within a smear of cul- tured parasites which had been allowed to develop into gametes or ookinetes. Female gametes are small, pigmented cells which have emerged from the ery- throcyte and which hybridise with the parasite- specific SSu rRNA probe (Fig. 3. panel la). Female gametes, identified by these criteria, showed a strong signal following hybridisation with the Pbs21-specific probe (Fig. 3. panel lb). At later stages of development, Pbs21 mRNA was also detected at high levels in immature and mature ookinetes (Fig. 3. panel 2b and 3b respectively). The overall distribution pattern of Pbs21 mRNA throughout the cytoplasm of female gametes and ookinetes resembled that observed in

J

Fig. 3. Expression of Pbs21 mRNA in sexual-stage parasites. In situ hybridisation to P. berghei gametes (Fig. 3.1) and ookinetes (Fig. 3.2). Parasites were identified by the signal obtained following hybridisation with a SSu rRNA-specific probe which was directly labelled with fluorescein (panel a). Expression of Pbs21 mRNA was visible following hybridisation with a digoxigenin-labelled Pbs21-specific probe, detected with a rhodamine-conjugated anti-digoxigenin antibody (panel b) in gametes (gam) but not asexual-stage parasites, including trophozoites (t). nucleus, N. Scale bar, 5 ~m.


female gametocytes and, in addition, Pbs21 mRNA appeared to be present at similar low levels within the nucleus.

4. Discussion

Pbs21 is capable of invoking an effective immune response within mice which can block the development of P. berghei parasites in the mosquito vector. The parasite does not express this antigen during the vertebrate stages of its development and only initi- ates the synthesis of Pbs21 protein following the activation of female gametes. We have shown, by in situ RNA hybridisation, however, that Pbs21 mRNA can be detected within mature unactivated female gametocytes developing within the vertebrate host. The results reported here, therefore, support the sug- gestion [15] that the parasite is able to synthesise high levels of Pbs21 protein rapidly during gametogenesis as a result of the pre-accumulation of a large pool of Pbs21 mRNA within gametocytes, which is immediately available for translation.

A number of possible mechanisms may operate within the female gametocyte to ensure that Pbs21 protein is not expressed until transmission has taken place. Such mechanisms may involve an enhance- ment of the rate of turnover of newly synthesised Pbs21 protein in gametocytes or, alternatively, more direct post-transcriptional mechanisms including re- pression of the translation of Pbs21 mRNA as a result of the retention of mRNAs in nuclei as unprocessed precursors, or storage of the mRNA in subcellular compartments. We have looked for evidence of localisation of Pbs21 mRNA at the sub-cellular level, therefore, using the technique of in situ RNA hybridisation and have shown that Pbs21 mRNA does not appear to be retained in the nuclei of sexual-stage parasites at any stages of their development but is distributed throughout the cytoplasm.

The recent discovery that the expression of different forms of the small subunit of ribosomal RNAs is regulated in a stage-specific manner [20,21] has also raised the possibility that mosquito-stage proteins, including Pbs21, may be translated preferentially by the ribosomal forms expressed by the parasite following transmission. Using probes which will discriminate the patterns of expression not only of

individual RNAs but also of closely related members of gene families, such as the genes encoding stage- specific rRNAs, it will be possible to determine whether the expression and translation of sexual-stage mRNAs within individual parasites correlates with transcription and processing of different forms of SSu rRNA. In addition, extension of this technique to the electron microscope level will allow us to observe directly whether translation of mRNAs correlates with their association with ribosomes containing particular forms of SSu rRNA.

Acknowledgements

The authors would like to thank Drs. Andy Wa- ters and Chris Janse for their very helpful advice and comments on the manuscript, Dr. David Tannahill for advice on the methodology, Drs. Theresa Ani- mashawa, Geoff Butcher and Ms. Louise Williams for their help in the preparation of parasites, Dr Furio Spano for providing the Pbs21 plasmid, and, in particular, Dr. Guy Barker for his many contribu- tions throughout this study. This work was supported by the European Commission Research Program, STD 3, and the Medical Research Council of Great Britain.

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in situ detection of pbs21 mrna during sexual development of plasmodium berghei

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