(Received for publication, May 28, 1996, and in revised form, October 9, 1996)
From the Department of Parasitology, University of
Leiden, Postbus 9605, 2300RC Leiden, The Netherlands,
Department
of Cytochemistry and Cytometry, University of Leiden, 2333 AL Leiden,
The Netherlands, and ** Department of Biology, Imperial College,
London SW7 2BB, United Kingdom
Malaria parasites (Plasmodium spp.) differentially express structurally distinct sets of rRNA genes in a stage-specific manner. The four rRNA genes of the rodent malaria parasite, P. berghei, form two classes of 2 units that are genetically unlinked and termed A-type and S-type. Through Northern analysis and in situ hybridization, expression of the units was demonstrated in synchronized parasite preparations covering the developmental pathway from the initiation of the blood-stage asexual cycle to the production of mature ookinetes. A-type units were transcribed in direct response to cell growth in bloodstage asexual parasites yet were differentially regulated during male (inactive) and female (active) gametocytogenesis. S-type expression was not confined solely to the mosquito stages and exhibited a finite period of expression in a subset of bloodstage trophozoites that was significantly elevated in gametocyte-producing parasites. Unlike in the human parasite, P. falciparum, there was no evidence for accumulation of precursor forms of the S-type transcripts in gametocytes. No significant rRNA transcription was observed in cultured, fertilized ookinetes until ~20 h of development when S-type transcription was initiated. The results further demonstrate that in Plasmodium the expression of the different rRNA units is linked to developmental progression but in a species-specific manner.
One of the remarkable features of Plasmodium parasites
is the developmentally regulated ribosome characterized by
transcription in a stage-specific manner of discrete repertoires of
unlinked nuclear rRNA genes, now termed A-type (from
sexual) and S-type (from
porozoite) (1). The
exceptional pattern of transcription of these genes was initially
discovered in the rodent parasite, Plasmodium berghei, where
it was broadly demonstrated that the asexual blood stages transcribe
the A-type units, whereas the S-type units are transcribed in the
mosquito stages of the parasite, including the sporozoite (2). A
similar pattern of rRNA unit transcription has been confirmed for every
species of Plasmodium investigated (3-5). Subsequent work
in different parasite species demonstrated that the switches in rRNA
unit transcription were linked to parasite development associated with
transitions between the vector and host. Thus, it was shown in the
human parasite, Plasmodium falciparum, that the switch from
A- to S-type transcription occurred during gametocytogenesis and zygote
formation and involved accumulation of S-type rRNA precursor forms (6).
In P. berghei, the switch back to A-type transcription
occurred early in the liver stages following invasion of cultured
hepatocytes by sporozoites (7). Recent work in Plasmodium
vivax has shown that the transition to S-type rRNA occurred much
later in the period of development within the mosquito, at the point in
oocyst development when sporozoites were being formed and contrasting
with the observations in P. falciparum (5).
Lately, a third type of developmentally regulated nuclear rRNA gene has been demonstrated in P. vivax, a finding that may prompt a re-evaluation of all previous data. Termed O-type, their transcription was first detectable in ookinetes, and their period of maximum transcription was in oocysts separating those of the A- and S-types. O-type genes are structurally significantly different from both the A- and S-type. Transcripts of the O-type appeared to be confined to the oocyst structure and were not detected in salivary gland sporozoites.1
These observations and the fact that the O-type has not yet been demonstrated in P. berghei indicated that there may be species-specific features of the switch and the types of genes involved. The four rRNA units in the nuclear genome of P. berghei are currently divided into two pairs representing the A-type (A and B units) and S-type (C and D units)(8, 9), which somewhat simplifies analysis. We present here a description of the steady-state transcriptional activity of these stage-specific units through the use of probes directed against the relatively rapidly evolving A and C unit external transcribed spacer (ETS)2 regions. We found that transcription of the P. berghei nuclear rRNA genes was also developmentally regulated. Specific and unexpected periods of rDNA unit transcription were observed, demonstrating a complex pattern of transcriptional regulation that could be linked to the various differentiation pathways of the parasite.
The following cloned lines of the ANKA strain of P. berghei were used in this study: 8417 (gametocyte producer); clone 2.34 (producer); 8458 (low producer); and clone 2.33 (nonproducer). In addition, the nonproducer clone 1 of the K173 strain was used (10).
Production of the Various Stages of the ParasiteSynchronous asexual blood stages were obtained from infections in rats that were synchronized as has been described (11-13). Parasites synchronized in this way typically have a window of development no greater than 3 h. Blood containing ring forms was collected from the rats, and leukocytes were removed as described and incubated under standard in vitro culture conditions (11) for 21-24 h to allow the development of the rings into mature schizonts. From these cultures, samples were taken at the appropriate time points for RNA extraction (see below).
Immature gametocytes were purified from blood collected from rats with synchronous infections as described (14). These immature gametocytes were incubated for a period of 6 h under standard culture conditions (11) for complete maturation. The purified gametocyte preparation consisted of 95% male and female gametocytes in a 1:1 ratio mixed with 5% mature schizonts and ring forms.
Zygotes and ookinetes were obtained from in vitro cultures as described (15). In this study, we used two different sources of gametocytes to start the cultures: 1) purified gametocytes, which were collected as described above (in these cultures, normally 50-70% of the females were fertilized, and there was a 5% schizont contamination (10)); and 2) infected blood obtained from mice with a high proportion of gametocytes (16). From this material, ookinete smears were prepared for in situ hybridization or were purified from unfertilized gametes, young gametocytes, and trophozoites by density gradient centrifugation, resulting in ookinete preparations consisting of mature ookinetes, uninfected cells, and ring forms.
Nucleic Acid Techniques: RNA ExtractionRNA was prepared from purified parasites as reported previously (10). RNA was fractionated in formaldehyde containing agarose gels and blotted to nylon membrane (Hybond N+, Amersham Corp.) according to standard protocols (17).
Definition of the rRNA Units and Probes Used in This StudyThe four rRNA gene units of P. berghei were
originally defined as A-D according to their size upon digestion of
genomic DNA with EcoRI (8). This definition has survived the
discovery of the specific developmental transcription of the different
units, which are now defined as A (sexual vertebrate
host), S (
porozoite), and more recently, O
(
ocyst). This work confirms what was evident from the
early restriction mapping that the four units form two groups of two
units, currently termed A and S, and the individual units will continue
to be referred to as A, B, C, and D. As discussed, it is not yet
possible to distinguish between the C and D units, although restriction
analysis (8, 18) and preliminary sequence data3 indicate that this should be
possible. The S units may yet have to be further redefined into an O
and S type. New probes generated during the course of this study are
referred by numerical designation according to use in the laboratory
with lettering A or S according to the type of unit they were derived
from and detect.
The probes were derived by subcloning or PCR
amplification from the following two genomic DNA clones that have been
described previously (8, 19). A-unit probes were derived from clone pPbS5.6 (8), a 5.6-kilobase pair EcoRI/HindIII
fragment of P. berghei genomic DNA, which contains the
A-unit SSU gene and 3.0-kb of upstream sequence. S-unit
probes were derived from clone pPbSL8.8 (19), an 8.8-kb
EcoRI/HindIII fragment of the C P. berghei genomic DNA that contains part of the C-unit
LSU gene, the 5.8S gene, the SSU, and ~1 kb of
sequence upstream of the SSU. These clones and the regions used in this
study as probes are illustrated in Fig. 1.
Oligonucleotides used in the PCR amplification reactions were: 365R,
5-TAA TAC GAC TCA CTA TAG GGA GAG ATC AAC CAG GTT-3
, which
corresponds in an antisense fashion to the 5
end of the mature 18 S
unit of the A and C rDNA units but also includes the T7 RNA promoter
for the optional generation of antisense RNA probes (this was used for
the amplification of ETS probes from both the A and C units); 38A,
5
-TAT GCA TCA TAA TAA AGA-3
, which corresponds in a sense fashion to
position
395 with respect to the 18 S gene of the A
unit4 and when used with 365R, generated
the 101A probe of 438 nt; and PUC R reverse sequencing primer, 5
-AGC
GGA TAA CAA TTT CAC ACA GGA-3
, was used with 365R to amplify 99S, an
~1-kb probe containing sequences homologous to the C unit ETS region.
PCR conditions were 4 cycles of 94 °C for 30 s, 40 °C for
30 s, and 72 °C for 1 min, followed by 25 similar cycles but
with an elevated annealing temperature of 54 °C. The Taq
polymerase used in these studies was purchased from Life Technologies,
Inc. The antisense oligonucleotides TM4 and TM3 are specific for the A-
and S-type SSU rRNA, genes, respectively and have been described before
(2). Final wash conditions for these probes was at
Tm (melting temperature)
5 °C in 3 × SSC, 0.5% SDS (1 × SSC is 0.3 M NaCl, 0.03 M sodium citrate).
Clone 315A is an EcoRI/NsiI subclone from pPbs5.6 (18) encompassing 2.6 kilobase pairs of the region upstream of the mature SSU A unit (Fig. 1). The clone of the P. falciparum A-type SSU rRNA gene has been described before (3).
Probes 104S and 105A are PCR-generated probes specific for the internal
transcribed spacer region 1 of the C and A units, respectively, and are
shown in Fig. 1. Final wash conditions for all complex probes was
0.1 × SSC, 0.5% SDS at 65 °C. Templates containing a 3 T7
RNA polymerase initiation site for the synthesis of antisense RNA
probes by in vitro transcription were generated by two
rounds of PCR amplification as follows.
A template for the transcription of a P. berghei
rRNA-specific probe was generated by amplification of a 190-nucleotide
fragment of clone pPbS5.6, containing P. berghei A SSU rRNA,
using the 5 primer CTTAACCTGCTAATTAGC in conjunction with the 3
primer GACTCACTATAGGGCCACCGTTCCTCTAAG, which includes 15 nucleotides corresponding to the T7 RNA polymerase initiation site. PCR
amplification was carried out in the presence of the 3
primer alone
for 5 cycles at 94 °C for 1 min; 36 °C for 1 min; and 72 °C
for 1 min; then, in the presence of both primers, for 30 cycles at an
annealing temperature of 48 °C. To complete the T7 RNA polymerase
initiation site and add 10 bp of protecting sequence, a second round of
amplification was carried out under the same conditions, using 0.5 µl
of the first amplification reaction mix in the presence of the same 5
primer and the 3
primer ACAAGCTTCTAATACGACTCACTATAGGGC.
A template for the transcription of a P. berghei rRNA
S-ETS-specific probe was generated in the same way by amplification of
a 246-bp fragment of the C-unit ETS region within clone pPbSL8.8 in the
presence of the 5 primer ATTCACTTCCGTAATTTG and, during the first
round of amplification, the 3
primer GACTCACTATAGGGCTCATTTACCGTGATC. The annealing temperatures during the first round of amplification were
5 cycles at 32 °C and 30 cycles at 42 °C, and during the second
round were 5 cycles at 36 °C and 30 cycles at 46 °C. A template
for the transcription of a P. berghei rRNA A-ETS-specific probe was generated by amplification of a 438-bp fragment of the A-ETS
region within clone pPbS5.6 in the presence of primers 365R and 38R as
described above.
Chromosomes of the different strains and clones of P. berghei were fractionated by pulsed field gel electrophoresis, transferred to nylon, and hybridized using conditions described previously (20).
In Situ Hybridization to Malaria ParasitesThe methodologies describing the preparation of P. berghei parasites and suitable RNA and DNA probes for in situ RNA hybridization and their application have been described thoroughly elsewhere (21-23). Probes were labeled either through the direct incorporation of fluorescein-UTP or the incorporation of digoxigenin-UTP/dUTP as appropriate and detection with fluorochrome-linked specific antibodies. All chemicals and reagents for these processes were purchased from Boehringer Mannheim. The method of visualization used to generate the data shown is indicated in the text and figure legends.
The reader is referred to "Materials and Methods" for
the definition of the RNA units. The ETS regions of the A and C rRNA units were isolated from the genomic clones described previously (18)
by a combination of PCR and subcloning procedures. These and other
probes used in this study are illustrated in Fig. 1, which is a schematic representation of the A and C units showing the
gene units and the ETS and internal transcribed spacer regions (adapted
from Ref. 19). These probes were used to establish the chromosomal
location and sequence type of the units and their patterns of
transcription in the various stages of the life cycle reported here. A
probe for the small subunit rRNA gene hybridized to chromosomes 5, 6, 7, and 12, showing that the four rRNA units are unlinked in the genome.
The A-type ETS probes (101A and 315) hybridized only to chromosomes 7 and 12, whereas the S-type ETS probe (99S) hybridized to chromosomes 5 and 6 (Fig. 2). This demonstrated that probes directed
against the ETS regions of the A and C units were type specific,
consistent with the observed similarity of respective restriction maps
of the four units (8, 24). The sequence of these regions will be the
subject of another report that identifies and characterizes the
transcription start site of the rRNA gene
units.5 The internal transcribed spacer
region 1 probes (Fig. 1) have the same A- or S-type specificity (data
not shown).
Transcriptional Activity of the rRNA Genes in Asexual Blood Stages and Gametocytes
The typical rDNA unit is transcribed as a single
large primary transcript, which is then processed through a series of
characteristic steps to generate the three mature rRNA species of SSU
rRNA (~2050 nt for Plasmodium), 5.8S (~155 nt), and the
(naturally nicked in P. berghei) LSU rRNA (~800 and 3000 nt)(8, 18). The ETS probes used in this study will, therefore, detect
the primary transcript, and any secondary processed precursor species
that contain the ETS but will not hybridize directly to the mature rRNA
species. The Northern and in situ hybridization analyses
demonstrated that the ETS probes hybridized only to precursor rRNA
species and that these were confined to the nucleus (Fig.
3). The processing of rRNA precursor transcripts is a
rapid process, and the RNA species have extremely short half-lives.
Nuclear run-on analyses using blood-stage parasites confirmed that the
A-type units are actively transcribed in these cell types (data not
shown). Probes detecting these intermediates could, therefore, be used
to measure steady-state rRNA unit transcription. This was performed
using synchronous populations of distinct clones of P. berghei, which either could or could not produce gametocytes.
Transcription of the rRNA units in
blood-stage parasites. A, Northern analysis of A-type
transcription. RNA was isolated from synchronous populations of clone
8417 (gametocyte producer) and from clone 233 (gametocyte nonproducer)
and subjected to Northern analysis. Northern blots of the isolated RNA
were sequentially hybridized to the A-type ETS probes 315A and 101A and
to the oligonucleotide TM4. Lanes 1-5 contain RNA of clone
8417. Lane 1, 5 hpi (called ring stages); lane 2, 16 hpi (trophozoites); lane 3, 25 hpi (maturing schizonts);
lane 4, 28 hpi (mature schizonts); lane 5, purified gametocytes 35 hpi. Lanes 6-9 contain RNA of clone
233. Lane 6, 5 hpi; lane 7, 16 hpi; lane
8, 25 hpi; lane 9, 28 hpi. Diagrams of the morphology
of the different stages are placed above the appropriate lane of the
blot. The small forms illustrated beneath the schizonts in lanes
3 and 4 are gametocytes, which represent about 20% of
the population in this parasite clone. B, in situ RNA
hybridization analysis of A-type transcription. Left column, 4,6-diamidino-2-phenylindole-stained nuclei. Right column, in situ RNA hybridization of digoxigenin-labeled probe 101A to
asexual parasites. Hybridization was detected with fluoroscein-labeled anti-digoxigenin antibodies. 1, trophozoites; 2, schizonts of increasing maturity (increasing number of nuclei; 16n is
fully mature) and a unicellular merozoite (Mz);
3, a male (
) and female (
) gametocyte and a trophozoite (T). C,
Northern analysis of S-type transcription. The same blot used for the
previous hybridizations was sequentially hybridized to the S-type ETS
probe, 99S, and to the S-type specific oligonucleotide TM3 (data not
shown). Lanes 1-9 are as in A. D, in
situ hybridization of the digoxigenin-labeled S-type ETS probe,
99S, to synchronous preparations of clone 8417. The positive signals
were only observed in 16-hpi mononuclear parasites (trophozoites).
Left panel, 4
,6-diamidino-2-phenylindole-stained nuclei.
Right panel, in situ hybridization of 99S.
Four time points were investigated representing each of the morphologically distinct phases of the asexual blood-stage cycle (5 hpi, consisting of ring stages/young trophozoites; 16 hpi, old trophozoites; 25 hpi, mature schizonts/young gametocytes; and 28hpi, mature schizonts/mature gametocytes). RNA from equivalent numbers of parasites were loaded in each lane, which was quantitated by hybridization of an oligonucleotide (TM4) to the mature SSU rRNA species. In the gametocyte-producing clone (8417), the A-type units were transcribed throughout the asexual cycle, and the production of two major precursor species of 2700 and 2900 nt was observed hybridizing to probe 101A (Fig. 3A). A faint band of ~7.0 kb also hybridized to 101A and is interpreted as the size of the primary rRNA transcript of the complete unit or an early processing intermediate (data not shown). The 2900-nt precursor was only observed in the first 16 h after erythrocyte invasion, and both major precursors were equimolar during this period. However, the 2900-nt precursor was not observed during schizogony or in mature gametocytes. In the gametocyte nonproducing clone, the 2900-nt precursor is in the same parasite forms and to the same extent as in the producer clone (Fig. 3A, compare lanes 1, 2, 6, and 7). However, A-type transcription was barely observable in mature schizonts (Fig. 3A, compare lanes 3, 4, 8, and 9). We conclude that the 2700-nt precursor is produced by gametocytes, and these forms are responsible for the signal observed in the schizont samples taken from the gametocyte-producing parasite clone. Mature schizonts do not, therefore, transcribe rRNA genes. This pattern of transcription was observed using both 101A or TM4 (data not shown). Preliminary data indicated that the observed precursors consisted solely of the mature SSU and ETS regions.6
These conclusions were confirmed and clarified by in situ hybridization of 101A to blood-stage parasites, which demonstrated that transcripts containing the ETS region were confined to the nucleus, consistent with the exclusive nuclear processing of eukaryotic rRNA primary transcripts (Fig. 3B). A-type transcription was not observed in newly invaded rings (data not shown) but was observed in all asexual mononuclear stages from 5 hpi. Furthermore, A-type transcription decreased with increasing schizont maturity and was barely detectable in fully mature schizonts (16n) and released merozoites (1n). A-type transcription in gametocytes was shown to be sex-specific. Female gametocytes continued to transcribe the A-type units, whereas the males were silent (Fig. 3B).
The S-type units were transcribed in a phased manner in blood-stage parasites (Fig. 3C). S-type precursors of an estimated size of 3000 and 2650 nt, respectively were observed with a peak in production at 16 hpi, when all cells are maturing trophozoites. The same pattern of S-type precursors was observed in both gametocyte producer and nonproducer clones, although the level of transcription in the former was significantly higher (Fig. 3C). The level of S-type transcription appeared to vary between clones between 2- and 10-fold, as estimated by densitometry (Fig. 3C) and appeared to be consistently greater in producer clones. The larger precursor was more abundant than the smaller (ratio, 5:1). A third band of >10 kb was also visible, which is interpreted as being the full-length primary rRNA transcript of the complete unit comparable to the smaller >7.0kb transcript of the A-type unit. The transitory appearance of the S-type precursors indicated that they did not accumulate as steady-state precursor forms that had been observed in gametocytes of P. falciparum (4), nor was accumulation of the mature transcripts observed through hybridization to the S-type-specific oligonucleotide, TM3 (data not shown). Neither transcription of the S-type units nor accumulation of S-type transcripts in mature gametocytes was evident (Fig. 3C) in clone 8417 illustrated nor in rings, mature trophozoites, nor schizonts from independent nonproducer parasite clones, LK173 and 233 (see "Materials and Methods"; data not shown).
In Situ Hybridization of the S1ETS probe to synchronous blood-stage parasites confirmed that the peak of S-type transcription occurred at 16 hpi during trophozoite development. The peak appeared to be restricted to a subset of mononuclear cells (<1%). As in Northern analysis, neither S-type transcription nor accumulation was observed in mature gametocytes (Fig. 3D). It is not yet possible to distinguish between 16-hpi trophozoites, which will form schizonts, and those committed to sexual differentiation.
Transcription of the Nuclear rRNA Gene Units during Sexual Development and Zygote DevelopmentUsing the ETS-specific probes,
A-type and S-type transcription was examined in mature gametocytes,
zygotes, and developing ookinetes (Fig. 4). A-type
precursors were still abundant in mature female gametocytes (Fig.
4A, right panel). After fertilization, it appeared that
transcription of the A-type units rapidly decreased. In the ookinete
preparations, which were derived from purified gametocytes, A-type
precursors were hardly visible at 2 and 14 h after fertilization.
The strong hybridization at 22 h is due to the presence in this
particular preparation of ookinetes of large numbers of asexual ring
forms that produce significant amounts of A-type precursors (Fig.
2B). In situ hybridization analysis confirmed
that A-type transcription was down-regulated in zygotes and ookinetes
during this period and that the nuclear A-type signal resulted solely
from asexual parasites present in the culture (Fig. 4B).
In contrast to the rapid decrease in A-type transcription, S-type transcription was up-regulated during ookinete development (Fig. 4A, left and center panels). In mature gametocytes, little if any S-type transcription was seen. S-type transcription could be detected 2 h after fertilization and dramatically increased between 14 and 24 h in the maturing ookinete. Four S-type rRNA precursor bands were observed in ookinetes, the two observed in asexual parasites (3000 and 2650 nt) and two larger forms (3900 and 3400 nt). The 2650-nt precursor was more abundant than the 3000-nt transcript in contrast to the transcription of the S-type units in the blood-stages, where the relative abundance of these two precursors was reversed (Fig. 2C). In situ hybridization analysis confirmed that the S-type transcription could be detected in the nucleus of about 10% of 20-h ookinetes and in >70% of mature (>24-h) ookinetes. S-type transcription was not detected with this method in 2-h zygotes or in 4-, 6-, 12-, and 16-h ookinetes (Fig. 4B and data not shown).
The pattern of rRNA gene transcription in Plasmodium parasites is complex. This is due not only to the presence of different types of rRNA gene units (A- and S-type) that are transcribed in a stage-specific manner (2-5) but also the presence of multiple and unlinked copies of each type. Each individual unit may be differentially transcribed during the progression of the life cycle, complicating the analysis. The pattern of transcription of the rRNA genes can be established relatively easily in the rodent model malaria parasite, P. berghei, because there are only two copies each of the A-type (the A and B units) and the S-type (the C and D units)(8), and the types can be defined by the ETS probes described here. These probes also further define the extent of pairwise homogeneity between the units. The mechanism that underlies the maintenance of this pairwise homogeneity of units on separate chromosomes involves some level of gene conversion (25), yet it must also discriminate between and maintain the different types.
In Plasmodium, rapid growth periods result in the formation of relatively stable parasite forms (mature schizonts/merozoites, gametocytes, and sporozoites) that only undergo further differentiation after a specific transition, e.g. erythrocyte invasion or passage between the host and vector. The work presented here demonstrates that rRNA transcription in two of these stable forms, schizonts and male gametocytes, is down-regulated and that only the female gametocyte continued to transcribe the rRNA genes. The female gametocyte can accumulate maternal mRNA that is not translated until after fertilization (e.g. Pbs21 ookinete antigen gene (10, 21, 22)) and may, therefore, require protracted ribosome biogenesis during its development and may also accumulate ribosomes for use in the zygote, given the absence of appreciable rRNA transcription in P. berghei at that stage. Electron microscopic examination of mature male gametocytes suggested that ribosomes were undergoing visible degradation, which would be consistent with their cessation of rRNA gene transcription (26, 27). The steady state pattern of A-type rRNA precursors was complex and probably cell type-specific, implying that the female gametocyte uses a different rRNA processing pathway compared to asexual blood-stage forms, even if the difference only reflects the kinetic stability of the intermediates. Alternatively, the range of rRNA precursors may reflect two specific forms of A-type transcript, one each produced by the A and B units. The currently available probes cannot discriminate between the units, which appear to be highly similar (8).
In most organisms, transcription of rRNA genes occurs during the growth phase of the cell (28). This work has shown that transcription of the A-type rRNA genes of P. berghei is maximal in the rapidly growing blood-stage forms and, therefore, under a similar type of control. In addition, we have shown that at least one of the S-type units, normally mainly transcribed during oocyst growth (2), is also transcribed during the blood-stage development in a small subset of P. berghei trophozoites and to a greater extent in parasite clones that produce gametocytes. Bloodstage transcription of the S-type units may indicate a temporary requirement for S-type ribosomes during blood-stage development, implying the existence of a specific mRNA population or cell type. This subset of cells may be committed to sexual development, but in the absence of additional early markers of gametocyte development, this cannot be confirmed. Unlike the observations made in P. falciparum (4), however, the P. berghei blood-stage S-type transcription did not result in the accumulation of precursor forms in gametocytes; in addition, there was no evidence of mature S-type SSU rRNA in any blood-stage form. This species-specific S-type rRNA gene transcription may well reflect the marked difference in the kinetics of development and morphology of P. falciparum gametocytes (26), which take a minimum of 8 days to mature, whereas in other malaria species, gametocytogenesis is of a similar length to asexual blood-stage development. Alternatively, it is possible we observed a general transcriptional event that is so transient that at any time point of analysis, transcription appears to be limited to a subset of the synchronized populations that have a developmental window of about 4 h. The developmental timing of the switch between A- and S-type transcription in the mosquito also differs in P. vivax, where there is a lag phase between days 2 and 6 after infection where neither class of rRNA is detectable (5).
The recent report of a third type of developmentally regulated nuclear rRNA gene transcribed during this lag phase in oocysts of P. vivax1 raises the question of whether a similar type exists in P. berghei. This could only result from a subdivision of the very similar but distinguishable S-type C and D units because we have demonstrated that the A types are barely transcribed throughout the mosquito stages and are almost identical (2). Characterization of the differences between the C and D units will allow this question to be addressed.
In conclusion, these studies demonstrate that transcription of the
stage-specific rRNA genes of P. berghei is highly regulated and involves different periods of activation demonstrably linked, in
the case of the A-type, to periods of cell growth. In addition to this
general conventional pattern, it was observed that switching of
transcriptional activity between the two different rRNA gene types was
linked to the various differentiation pathways of the parasites.
Superimposed upon this general pattern of transcription were transient
bursts of rRNA transcription, which may yet prove to play a role in
parasite differentiation (see Fig. 5 for summary of
known transcription patterns of rRNA throughout the life cycle of
P. berghei). The general pattern is directly analogous to
the transcriptional activity of rRNA genes found in the well studied eukaryotic models. Typically, transcriptional regulation or rRNA units
in these models results from different types of competitive associations of both transcriptional promoter (e.g. SL1,
UBF, TBP, and REB1) and repressor proteins (e.g. Rb) with
both enhancer and promoter DNA elements (29, 30). These associations
can be affected by the phosphorylation status of protein factors (31). Additionally, the status of the chromatin and the phasing of
nucleosomes in the region of the rDNA units can influence transcription
(32, 33). All of these mechanisms may play a role in the regulation of
the individual rDNA units of Plasmodium, and their interplay may then control the relative expression of each unit. The work described here implies that the general transcription of the rDNA units
of Plasmodium will, however, be regulated in a similar
conserved fashion to those of other eukaryotes, and the particular
interest will lie in examining the supplementary mechanisms that effect the observed stage-specific control of rDNA transcription.
We thank Dr. Tom McCutchan for his continuing interest and advice.