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J. Biol. Chem., Vol. 276, Issue 25, 22638-22647, June 22, 2001
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From the
Received for publication, February 8, 2001, and in revised form, April 4, 2001
Unlike most eukaryotes, many apicomplexan
parasites contain only a few unlinked copies of ribosomal RNA (rRNA)
genes. Based on stage-specific expression of these genes and structural
differences among the rRNA molecules it has been suggested that
Plasmodium spp. produce functionally different
ribosomes in different developmental stages. This hypothesis was
investigated through comparison of the structure of the large
subunit rRNA molecules of the rodent malaria parasite,
Plasmodium berghei, and by disruption of both of the rRNA
gene units that are transcribed exclusively during development of this
parasite in the mosquito (S-type rRNA gene units). In contrast to the
human parasite, Plasmodium falciparum, we did not find
evidence of structural differences in core regions of the distinct
large subunit rRNAs which are known to be associated with catalytic
activity including the GTPase site that varies in P. falciparum. Knockout P. berghei parasites lacking
either of the S-type gene units were able to complete development in both the vertebrate and mosquito hosts. These results formally exclude
the hypothesis that two functionally different ribosome types distinct
from the predominantly blood stage-expressed A-type ribosomes, are
required for development of all Plasmodium species in the
mosquito. The maintenance of two functionally equivalent rRNA genes
might now be explained as a gene dosage phenomenon.
Ribosomes are essential cellular components that play a central
role in protein synthesis. It has been demonstrated that the ribosomal
RNA (rRNA) has an active role in the assembly, structure, and
interaction of the ribosomal subunits (1-3) and a direct role in
catalysis and accuracy of protein synthesis (4, 5). The rRNA molecules
present in eukaryotic ribosomes are identified by sedimentation
properties as 28 S (large subunit
(LSU)1), 18 S (small subunit
(SSU), 5.8 S, and 5 S. The genes encoding the first three rRNA
molecules are expressed from a single rRNA gene unit as one polygenic
transcript that is subsequently processed. In most eukaryotes,
100-10,000 identical copies of the rRNA gene unit are present
per haploid genome, clustered in tandem arrays. Because the rRNA
molecule is the catalytic center of the ribosome (1, 5, 6), their
sequence conservation presumably reflects functional constraints that
are required for optimal translational efficiency.
Unicellular apicomplexan parasites have superficially typical rRNA gene
units that are comprised of 18 S, 28 S, and 5.8 S genes, separated by
the internal transcribed spacer (ITS) regions and flanked by external
transcribed spacer (ETS) regions (7-11). However, in contrast to most
eukaryotes, numerous, but not all, apicomplexan species contain a
characteristically small number (two to seven) of structurally distinct
rRNA gene units that are unlinked in the genome (11-14). This
phenomenon has been characterized in the greatest detail in
Plasmodium spp. parasites that are responsible for malaria. In Plasmodium, and in contrast to other
apicomplexans that possess dispersed genes, these rRNA gene units are
transcribed stage-specifically, thereby reducing the total number of
gene units that are simultaneously active (15-19). Based on the
differences in expression pattern and nucleotide sequence of the rRNA
gene units the existence of three types of structurally different
ribosomes in Plasmodium has been postulated. The A-type
ribosomes are present in the liver and blood stages of the parasite,
and the O- and S-type ribosomes are the predominant types produced
during development in the mosquito (20). The presence of structural
differences among the rRNA molecules has led to the hypothesis that the
different ribosome types are also functionally different (15, 18, 19). Two observations support this hypothesis. First, the O- and
S-type rRNA genes of the human parasite Plasmodium vivax
encode SSU rRNA molecules that differ in core regions that are involved
in mRNA decoding and translational termination (20). Second, in the human malaria Plasmodium falciparum, A-type and S-type LSU
rRNA molecules differ in the core regions, and most marked is the
distinct GTPase domain in the A- and S-type molecules (18, 21). It is
currently a complete mystery why malaria parasites evolved this
atypical organization and structure of the rRNA gene units. It has been
postulated that these differences reflect the need for the parasite to
propagate in two very different environments, the vertebrate host and
invertebrate vector (15).
To investigate the possible existence of functionally different
ribosome types we analyzed the rRNA gene units of the rodent parasite
Plasmodium berghei, an established model malaria parasite. This parasite contains four distinct copies of the rRNA units (A-D)
(22) divided into the blood stage A-type (A- and B-unit) and S-type (C-
and D-units), which is transcribed mainly in the proliferative stages
in the mosquito (14, 15). It has been possible to distinguish the two
S-type SSU genes (C- and D-SSU) on the basis of sequence differences
reminiscent of the S- and O-types of P. vivax (20). However,
we demonstrated that the C-SSU gene is actually a chimera of the A- and
D-SSU, and no differences were found in the core regions of the A-, C-,
and D-SSU rRNA molecules (23) which might imply functional differences.
These results questioned whether two functionally different ribosome
types are required for development of P. berghei in the
mosquito as has been implied for P. vivax (20). Here we
investigated further the rRNA gene units by comparison of the sequence
and structure of the LSU genes of the different units. The sequence
information of the LSU and SSU genes allowed us to address possible
functional differences between the ribosome types by disruption of the
C- and D- (S-type) rRNA gene units. Unexpectedly, neither the
structural comparison nor the gene knockout studies revealed evidence
for the existence of functionally different ribosome types in P. berghei. Only one copy of the S-type genes, either the C- or the
D-gene, is sufficient for complete development of the parasite in the mosquito vector. A previously undescribed presence of the A-type rRNA
molecules in maturing oocysts may also account for the continued development of gene knockout (ko) parasites in the mosquito. Given the
similar phenotype of the C- and D-ko mutants we suggest that the
maintenance and transcription of the additional rRNA units in the
mosquito represent a gene dosage phenomenon that ensures efficient
progression of parasite multiplication in the relatively short lived
mosquito vector.
Isolation and Characterization of the LSU Genes of the Different
rRNA Gene Units
DNA fragments containing the LSU rRNA genes of the A-, C-, and
D-units were isolated for sequencing. The 5'-ends of the A- and C-LSU
rRNA genes present in clone pPbSL7.8 (22) and clone pPbSL8.8 (24)
respectively, were subcloned by PCR and sequenced completely. The
5'-end of the D-LSU rRNA gene was isolated from a library of
size-selected KpnI/HindIII restriction fragments (range 4-8 kb) derived from genomic DNA (clone 8417 of the ANKA strain) and ligated into the vector pBS/KS. The library was screened by
hybridization with oligonucleotide L87R (Table
I) specific for a conserved region at the
5'-end of the LSU rRNA genes (position 27-45). The final wash
was performed at 42 °C in 3 × SSC, 0.5% SDS. A plasmid
(referred to as pPbL4.8), containing the expected 4.8-kb
KpnI/HindIII fragment (11), was isolated, and the
insert was completely sequenced. The fragment contained 1.6 kb of the 3'-end of the ITS2 and 3.2 kb of the 5'-end of the D-LSU rRNA gene. We
were not able to clone the 5'-end of the second A-type, the B-LSU rRNA
gene. The 3'-end of the B-LSU gene (position 3062-3789), present on a
4.1-kb HindIII fragment (11), was ligated into vector
pBR322, resulting in the clone
pPbL4.1.2 This clone was
partially sequenced and contained 728 bp of the 3'-end of the B-LSU
rRNA gene plus 3.4 kb of downstream sequences. The 3'-ends of the A-,
C-, and D-LSU rRNA genes were isolated from a partial Sau3A
P. berghei genomic library in phage The secondary structure analysis of the P. berghei 5.8 S and
LSU rRNA molecules was inferred from comparative sequence analysis. The
eukaryotic LSU consensus sequence was calculated from an alignment of
23S-like rRNA sequences of 35 representative eukaryotic
species.3 The alignment was
made with the OmigaTM 1.0.1 software (Oxford Molecular Group). P. berghei positions different from the LSU consensus sequence were
checked for compensatory base changes by folding the P. berghei rRNA sequence into a three-dimensional structure similar
to the secondary structure model for P. falciparum (18).
Vectors
The construction of the vectors to disrupt the C- and D-rRNA
gene units was as follows.
Disruption Vector--
To create vector pMD207 (Fig.
1B), we first PCR amplified a
2038-bp fragment from the 5'-end of the C-SSU rRNA gene from plasmid pPbSL8.8 (24), using oligonucleotides L78R and 332R (see Table I; four
cycles annealing at 40 °C, 3-min extension followed by 30 cycles
annealing at 60 °C, 3-min extension) which introduced unique
EcoRI sites at either end of the fragment. This fragment was
cloned into the unique EcoRI site of vector pMD200 (25), resulting in vector pMD207. pMD200 contains the selectable marker cassette with the pyrimethamine-resistant DHFR/TS gene of P. berghei. Vector pMD207 has been used for disruption of both the C-
and D-rRNA gene units after linearization at the unique SpeI
restriction site. In pMD207, 21 bp of the C-SSU rRNA gene (nucleotides
2039-2059) are missing, resulting in the introduction of an incomplete
copy of the C-SSU rRNA gene after integration of pMD207 in the C- or D-rRNA gene unit (Fig. 1B).
Replacement Vectors--
Both vectors 387A and 395A (Fig.
1B) contain two fragments of the D-rRNA gene unit (24) on
either side of the selectable marker cassette present in vector
pDBDTm Transfection of P. berghei with Disruption and Replacement
Vectors
Transfection of P. berghei and selection of
transfected parasites were performed as described previously (26, 27).
Briefly, purified schizonts (108) of P. berghei
(ANKA strain, clone 15cy1) were transfected by electroporation with
either 40 µg of disruption vector pMD207 (after linearization at the
SpeI site) or 40 µg of the replacement vectors 395A or
378A (linearized with BamHI and EcoRI).
Transfected parasites were injected back into rats or mice, and
transfected parasites were selected by treatment of the animals with
pyrimethamine. Transfected pyrimethamine-resistant parasites were
cloned by the method of limiting dilution.
Genotype Analysis of Knockout Parasites
Infected blood was obtained from mice with a parasitemia between
5 and 40%, and leukocytes were removed using Plasmodipur leukocyte
filters (Eurodiagnostica). The parasites were either used for genomic
DNA isolation (28), chromosome separation by field inversion gel
electrophoresis (29), or parasites were used directly for PCR according
to a modified method from Snounou et al. (30).
105 parasites (obtained from lysed infected erythrocytes)
were washed with 250 µl of 1 × PCR buffer (Life Technologies,
Inc.), resuspended in 20 µl of 1 × PCR buffer, overlaid with
mineral oil, and incubated for 10 min at 100 °C. In each PCR 2 µl
of boiled parasite suspension was used as template. To demonstrate
correct integration of the vectors we used the oligonucleotides listed
in Table I. Disruption of the C-unit with pMD207 was detected with
oligonucleotides L270R and 328A (PCR-amplified fragment (PCRaf) of 3.0 kb); disruption of the D-unit was detected with L260R and 328A (PCRaf
of 3.0 kb). Wild-type (wt) C- and wt D-units were detected with L271R
in combination with L270R and L260R, respectively (both PCRaf sizes of
3.5 kb). Replacement of the C-unit with vector 395A was detected with
L270R and L393 (PCRaf of 3.2 kb); D-unit replacement was shown with L260R and L393 (PCRaf of 3.2 kb). In all of these PCRs 40 cycles, annealing at 55 °C, and 4-min extension were used. Wild-type
C-unit was detected with L263R and L264R (35 cycles, annealing at
55 °C, 2-min extension, PCRaf of 2.2 kb). For wt D-unit L265R and L266R (35 cycles, annealing at 55 °C, 2-min extension, PCRaf of 1.2 kb) were used. D-unit replacement with vector 387A was detected with
L392R and L393 (35 cycles, annealing at 55 °C, 1-min extension, PCRaf of 1.1 kb). Wild-type C- and wt D-unit were detected with L271R
in combination with L270R and L260R, respectively (see above).
For Southern analysis of genomic DNA of ko parasites three probes were
used. To demonstrate integration of vector pMD207, the C/D-ETS probe,
which is specific for the ETS region of the C- and D-units (probe 99S
(14)) was used. The C/D-ITS1 probe (see "Vectors"), which is
specific for the ITS1 region of the C- as well as the D-unit, was used
to show replacement by vector 395A. To visualize replacement of the
D-rRNA gene unit by vector 387A, probe D-ETS (see "Vectors")
specific for the 5'-ETS region of the D-unit was used. Hybridizations
were performed at 60 °C, and their final wash was at a stringency of
0.1 × SSC, 0.5% SDS, twice for 15 min at 60 °C.
Phenotype Analysis of Wild-type and Knockout Parasites
The presence of SSU and LSU rRNA transcripts of the different
gene units was analyzed by Northern analysis and RNA dot spot hybridizations. For oocyst RNA 15 midguts were dissected from anopheline mosquitos at day 10 after infection and collected in phosphate-buffered saline. For sporozoite RNA, salivary glands were
dissected from 50 mosquitos at day 20 after infection and collected in
phosphate-buffered saline. Directly after collection of the parasites,
RNA was isolated according to standard methods (28). For Northern
blots, RNA was fractionated in guanidine thiocyanate containing agarose
gels and blotted to nylon membrane (Schleicher and Schuell) according
to the protocol of Goda and Minton (31). For RNA dot spot blots,
aliquots of the RNA samples were denaturated and spotted on nylon
membrane (Schleicher and Schuell) by a dot slotter apparatus (Bio-Rad).
Blots were hybridized with oligonucleotides that are specific for the
rRNA genes of the different units (Table I). Hybridizations were
performed at 42 °C, and their final wash was at a stringency of
3 × SSC, 0.5% SDS, twice for 10 min at 42 °C. To determine
the relative amounts of the different rRNA transcripts, we measured the
intensities of hybridization signals using a PhosphorImager (Molecular
Dynamics) and the software ImageQuant 3.3 (University of Virginia,
ITC-Academic Computing Health Sciences). To correct for differences in
specific activity of labeled oligonucleotides, we simultaneously
hybridized these probes to plasmids containing the different rRNA
genes. The following plasmids were used (Fig. 1A for the
restriction fragments): pl351, containing the 7.8-kb EcoRI
fragment of the A/B-unit (pBbSL7.8 (22)); pl316, containing the 8.8-kb
EcoRI/HindIII fragment of the C-unit (pBbSL8.8
(11)); pl343, containing the 4.7-kb KpnI/HindIII
fragment of the D-unit (pPbS5.2 (23)); pl344, containing the 5.2-kb
KpnI/KpnI fragment of the D-unit (SSU to ITS1 region).
The phenotypes of the ko parasites were analyzed further using standard
technologies for determination of growth and development characteristics of P. berghei (32). Asexual blood stage
development and gametocyte production were determined in synchronized
infections in mice under standardized conditions (33). The gametocyte
conversion rate is the percentage of ring forms that develop into
gametocytes as determined by counting parasites in Giemsa-stained
slides. Ookinete production was determined in in vitro
cultures (34). The ookinete conversion is the percentage of female
gametocytes that develop into mature ookinetes in culture. In each
experiment the gametocyte and ookinete conversion rates have been
determined in two independent mice/cultures. Oocyst and sporozoite
development of ko parasites was analyzed in Anopheles
stephensi mosquitos after feeding on infected C57BL/10 mice.
Mosquitos were fed on days 3 and 4 on mice with a parasitemia of 1-7%
resulting from infection on day 0 with 107 parasites. In
each experiment mosquitos were fed on two mice, and 50-175 engorged
females were collected from each mouse. Mosquitos were kept for 20-32
days at a constant temperature of 21 ± 0.5 °C. At different
time points after infection, midguts of mosquitos were dissected for
monitoring oocyst development and RNA isolation. The mean number of
oocysts/mosquito was determined by counting the number of oocysts at
day 10 after infection in 30-50 mosquitos. The mean size of oocysts
was determined at day 10 as follows. Oocysts from three mosquitos
(80-120 oocysts/midgut) were photographed using a light microscope at
a magnification of ×400. From these images the size (diameter) of
50-160 oocysts was measured. The infectivity of sporozoites was tested
by feeding 25-50 infected mosquitos on naïve mice at day 20 after the infectious blood meal. In each experiment two mice were
infected, and the parasitemia in these mice was monitored by
examination of Giemsa-stained slides.
Comparison of the 5.8 S and LSU Genes of the Different rRNA Gene
Units--
The nucleotide sequence of the SSU genes of the A-, C-, and
D-units of P. berghei has already been published (15, 23, 35). The complete nucleotide sequences of the remaining 5.8 S and LSU
genes of these units are reported here. The sizes of these genes and
the identities between the genes of the different units are shown in
Table II. Although we have only sequenced
the ETS region (36) and 728 nucleotides of the 3'-region of the LSU of
the B-unit, all evidence indicates that the sequences of the A- and
B-units are identical. This is based on the identical restriction maps
of the A- and B-gene units (Fig. 1A), the comparable sizes
of the ITS1 and ITS2 as judged by PCR, the identical sequence of the
ETS region and of pPbL4.1 encoding the 3'-end of the B-LSU gene (11)
with that of the A-LSU gene (this study), and the comparable
hybridization intensities of A/B-unit-specific probes/oligonucleotides (Table I) to chromosome 7 and 12 (Fig. 1D and data not
shown).
A secondary structure analysis of the 5.8 S and LSU rRNA molecules of
the 3 units predicted the presence of all six expected domains that are
found in the conserved secondary structure model proposed for the 5.8 S/LSU rRNA hybrid of all eukaryotes (37, 38). The 5.8 S RNA molecules
of the A- and C-unit differed at the 3'-end in a manner covariant with
differences in the 5'-region of the LSU molecules (data not shown) as
observed for P. falciparum (18). This covariance between
these regions of the 5.8 S and LSU genes maximizes the specificity of
base pairing between the 5.8 S and LSU molecules of the same unit.
Sequence differences between the LSU RNA molecules of the different
units were compared with the standard consensus secondary structure
model for the LSU molecule of eukaryotes (38). From this comparison it
appeared that the majority of sequence differences occurred in the
variable regions of the eukaryote-specific LSU molecules (data not
shown), whereas in the core regions only 26 out of 1224 positions were different from the eukaryotic consensus sequence (Table
III).
If sequence differences between the core regions of different rRNA
molecules result in the presence of functionally different ribosome
types in Plasmodium, it could be expected that those sequence differences would be conserved between different
Plasmodium species. Therefore we compared differences
between the core regions of the A- and S-type LSU genes of P. berghei with the published differences between A- and S-type LSU
genes of P. falciparum (18). P. falciparum
maintains unique sequence differences in the core region of the LSU
gene associated with GTPase activity. The primary structure of this
region of the S-type LSU gene of P. falciparum more closely
resembles that of a bacterial GTPase center. In P. berghei
we found no differences in the GTPase region between any of the LSU
genes. They were identical to that of the A-unit of P. falciparum and show a standard eukaryote sequence and structure (position 1051-1108:
GTAGGACGGUGGUCAUGGAAGUCGAAAUCCGCUAAGGAGTGTGTAACAACTCACCTAC; underlined positions are variant in the P. falciparum
S gene). In the other LSU core regions all variant positions of the
P. berghei LSU were more similar to the A-unit of P. falciparum. In 7 out of 11 positions where P. falciparum A- and S-LSU genes differed, all three LSU genes of
P. berghei units were identical to the A-gene of P. falciparum, and 8 of the 11 P. berghei positions fit
the eukaryotic consensus sequence (Table III, bottom section). The A-
and S-type genes of P. berghei differ only at five positions in their core regions. Again however, these differences are not conserved in Plasmodium because the A- and S-type genes of
P. falciparum are identical at those positions (Table III).
Thus sequence differences between the core regions of the LSU genes of
A- and S-type units are a species-specific phenomenon.
19 positions in the core regions which differed from the eukaryotic
consensus sequence were conserved among all five Plasmodium LSU genes. 13 of these positions were expected to be engaged in the
formation of a bp, and these were found to be associated with compensatory changes that maintained the consensus structure (10 of 13)
or converted nonstandard Watson-Crick bp to standard ones (3 of 13)
(Table III, top section). Therefore, the typical differences from the
eukaryotic LSU rRNA core sequence found in Plasmodium are
supported by compensatory base changes that maintain known secondary
structure. The remainder of variant positions is species-specific and,
therefore, does not support the notion that stage-specific expression
of rRNA is associated with the production of functionally distinct ribosomes.
Generation and Isolation of Knockout Parasites with a
Disrupted/Replaced C- or D-rRNA Gene Unit--
To study possible
functional differences between the C- and D-rRNA molecules, we
generated ko parasites with a disrupted or replaced C- or D-gene unit.
To knock out the gene units, three different vectors were used (Fig.
1B). The disruption vector pMD207 contained an incomplete
copy of the C-SSU rRNA gene as target sequence. A single crossover
between the SSU rRNA sequences present in pMD207 and in the genome was
expected to disrupt the C- and/or D-rRNA gene unit as shown in Fig. 1
(Cdis and Ddis in Fig. 1C). In four independent experiments
we transfected parasites with vector pMD207 and obtained
pyrimethamine-resistant clones. In all experiments we obtained
parasites with a disrupted C- or D-rRNA as demonstrated by PCR. We
selected two C-disruptant (Cdis1, Cdis2) and two D-disruptant (Ddis1,
Ddis2) clones from independent experiments for further analysis.
Correct integration of vector pMD207 in the C- or D-rRNA gene unit in
the clones was confirmed by PCR (data not shown) and Southern analysis
of restricted genomic DNA and of separated chromosomes (Fig. 1,
D and E).
Analysis of both Cdis and Ddis parasites revealed that in a small
proportion of the ko parasites the wt genotype was restored. Reversion
to the wt genotype occurred through a reversal of the integration
mechanism leading to excision of the insertion vector. This reversion
phenomenon has been described before in ko parasites of P. berghei (39) and can complicate phenotypic analysis (see below).
Transfection with replacement vectors results in the specific and
irreversible inactivation of rRNA units, preventing restoration of the
wt genotype. Therefore, we also constructed the additional replacement
vectors 395A and 387A to knock out the C- and D-rRNA gene units. Vector
395A would delete the complete S-type 5.8 S gene, the ITS2 region, and
the 5'-end of the LSU rRNA gene. Although the vector was based upon
sequences of the D-unit it was anticipated that both the C- and D-units
could be interrupted because of the high homology between the units in
the regions chosen (Drep1 and Crep1 in Fig. 1C). We obtained
pyrimethamine-resistant parasite clones with a disrupted C- or D-rRNA
unit when vector 395A was introduced into the parasite, as demonstrated
by PCR. We selected both a C-replacement (Crep1) and a D-replacement
(Drep1) clone for further analysis and confirmed correct integration by
Southern analysis of restricted genomic DNA (Fig. 1E) and of
separated chromosomes (data not shown). Vector 387A was D-unit-specific and was designed to recombine with the unique part of the D-ETS region4 and part of the D-LSU rRNA gene. After integration
this vector would interrupt the D-ETS and LSU rRNA gene as well as
replace the SSU/ITS1/5.8 S/ITS2 region (Drep2 in Fig. 1C).
Transfection with vector 387A yielded parasites with a disrupted D-unit
as indicated by PCR. One D-replacement clone (Drep2) was selected for
further analysis. Correct integration of vector 387A in the D-rRNA gene
unit was confirmed by PCR (data not shown) and by Southern analysis of
genomic DNA (Fig. 1E) and of separated chromosomes (data not
shown). In all transfection experiments we never selected ko parasites
in which both the C- and D-rRNA gene units were disrupted and/or replaced.
The Presence of Transcripts of the Different rRNA Gene Units in
Wild-type and in Knockout Parasites--
The availability of the
sequence of the SSU and LSU genes of the different units allowed for
the design of a number of oligonucleotides specific for the different
rRNA molecules (Table I), which were used to analyze the relative
abundance of these molecules in the different developmental stages by
Northern analysis and dot spot analysis. As has been shown before,
mature S-type transcripts were absent during blood stage development of
wt parasites, and all SSU and LSU transcripts in asexual parasites and
gametocytes belong to the A-type (Fig. 2,
A and E). S-type transcription in wt parasites
starts in the developing ookinete and is up-regulated during oocyst
formation (14, 19) (Fig. 2A). We note that there is
significant A-type RNA present in mature oocysts and in sporozoites (Fig. 2, A and E). The mature LSU rRNA with a
size of about 4 kb is processed, resulting in two fragments: a small
5'-fragment of 800 bp and a large 3'-fragment of about 3 kb (40) (Fig.
2E). Because the mature S-type LSU rRNAs are either 153 bp
(D) or 242 bp (C) larger than those from the A-type units (Table II),
the corresponding 3'-fragments, resulting from the nick at 800 bp from
the 5'-end of the mature LSU rRNA molecule, have the same size
differences. Based on these size differences the A- and S-type LSU
rRNAs could be distinguished on Northern blots (Fig. 2B). This allowed for determination of the relative abundance of the A- and
S-type transcripts, and we calculated that mature oocysts contain A-
and S-type molecules in a ratio of 60% A to 40% S (Fig. 2B) This A/S-type ratio was confirmed by the determination
of hybridization intensity of different unit-specific oligonucleotides against both SSU and LSU in dot spot experiments. These dot spot experiments revealed also that the S-type transcripts present in both
mature oocysts and sporozoites consist of 3-5 times more molecules
originating from the D-unit than originating from the C-unit.
To determine whether knock out of the C- and D-rRNA units resulted in
the complete absence of rRNA transcripts of these units, RNA from
mature oocysts was analyzed (Fig. 2, D and E,
middle and bottom panels). All C- and D-ko
parasites were able to infect mosquitos, and oocyst numbers in
mosquitos were comparable to those in mosquitos infected with wt
parasites (see below and Fig. 3A). In the C-ko parasites no
C-unit-specific transcripts were observed, but D-rRNA was present.
Conversely, in all D-ko parasites, no D-rRNA was detectable, but
the C-transcripts were present. These results demonstrate that knock
out of the units completely blocked the synthesis of rRNA transcripts
from these units. A-type transcripts were present in oocysts of all ko
parasites (Fig. 2E, top panel). We found no
evidence that A-type transcription was up-regulated in oocysts and
sporozoites of the ko parasites (results not shown).
Phenotype Analysis of Knockout Parasites with Disrupted or Replaced
C- or D-rRNA Gene Unit--
Asexual blood stage development of all ko
parasites was comparable to wt parasites as determined by the length of
the asexual cycle (22-24 h), the number of merozoites/schizont (16 to
24), and the multiplication rate in synchronized infections in mice (results not shown). Also, the development of gametocytes and ookinetes
was not affected in all ko parasites (Fig. 3A). This lack of
an effect on blood stage and ookinete development is consistent with
the observations that S-type expression mainly takes place during
oocyst development in the mosquito and starts in the (nearly) mature
ookinetes. However, oocyst and sporozoite development in the ko
parasites was barely affected by the absence of transcripts of the C-
or D-rRNA unit. All ko parasites produced normal numbers of oocysts and
were able to produce infectious sporozoites (Fig. 3A). We
only observed a small, but significant and reproducible, difference in
the size of the oocysts of these parasites, indicating a slight
retardation of the growth of oocyst of the ko parasites (Fig.
3A). Only in parasites of Ddis1 did we observe a stronger inhibition of oocyst development (Fig. 3B), resulting in the
absence of sporozoites in day 10 oocysts in three independent
experiments. Surprisingly, the D-ko parasites with the same genotype
obtained in the second transfection experiment (Ddis2) showed a less
pronounced growth inhibition, and these parasites produced infectious
sporozoites comparable to the other C- and D-ko clones (Fig.
3A). This result suggests that the greater retardation of
oocyst growth of Ddis1 parasites resulted from not only the lack of
D-unit transcripts but also from an additional defect. The number of
sporozoites at day 20 in salivary glands in Ddis2 (11,000-22,000) and
Cdis1 (12,000-20,000) was comparable to that of wt parasites
(17,000-25,000), and sporozoites of all ko clones were infective to
mice. After transmission of the ko parasites through the mosquito, the
genotype of all ko parasites transfected with the replacement vectors
was the same as before transmission (Fig. 1F). In a few
experiments of the ko clones transfected with disruption vector pMD207
we observed a contamination of parasites with the wt genotype after mosquito passage (Fig. 1F). Those parasites arose as a
result of reversion events that can occur using this disruption vector (see above). This population of ko parasites containing parasites with
the wt genotype was treated with pyrimethamine during blood stage
development in mice, which resulted in the removal of wt parasites from
the ko population. This result proved that the wt parasites present in
these populations arose as a result of reversion events. The production
of infectious sporozoites in the ko clones demonstrates that the
presence of two S-type genes is not essential for the complete
development of P. berghei in the vertebrate host and in the
mosquito vector.
The function of the ribosome appears to be inextricably linked
with the conformation of the ribosomal RNA molecules (41). Cells of
most organisms contain homogeneous populations of ribosomes with regard
to the structure and function of rRNA molecules. Some, but not all,
apicomplexan parasites are an exception and produce ribosomes with
structurally distinct rRNA molecules encoded by single copy gene units
(7-11). This distinction is magnified in Plasmodium through
the stage-specific expression of the different rRNA genes (14, 18, 20).
Based on the structural differences in the core regions of the rRNA
molecules, the existence of three different ribosome types in
Plasmodium has been suggested: A-type ribosomes present
during development in the liver and the blood of the host and O- and
S-type ribosomes present during development in the mosquito vector. It
has been suggested that the maintenance of functionally different
ribosome types provides Plasmodium with a unique
post-transcriptional control mechanism to regulate gene expression (15,
19, 24). The expression of different ribosome forms could be a major
step in the commitment of the parasite to a change in life cycle stage
by influencing the population of messenger rRNAs (mRNAs) that are
translated. Alternatively, through maintenance of functionally
different ribosome types Plasmodium might be able to
optimize protein synthesis during growth in the different environments
of the host and the mosquito vector. Finally, structurally distinct
ribosomes may simply result from the fact that different segments of
the genome are accessible to the transcriptional apparatus during
different stages of the life cycle. Direct evidence for the existence
of functional differences among the three ribosome types of
Plasmodium is lacking. The most direct demonstration that
structural differences of the rRNA molecules might influence protein
synthesis is the observation of the different abilities of the GTPase
site of the A- and S-type LSU molecules of P. falciparum to
influence the growth of transformed Saccharomyces
cerevisiae expressing chimeric
yeast/Plasmodium LSU molecules (21).
Through a comparison of the two SSU rRNA genes of P. berghei
which are expressed during development in the mosquito (S-type rRNA
genes), no evidence was found for structural differences in their core
regions. This observation and the fact that both genes are expressed
simultaneously during the growth of oocysts questioned the existence of
two different ribosome types (O- and S-type) in the mosquito stages of
this parasite (23). The analysis of the primary structure of the LSU
rRNA genes of the C- and D-unit presented in this paper also
demonstrates that there is no structural basis for a distinct
classification of ribosomes containing C- or D-rRNA molecules which
might be related to known functional differences. The secondary
structures of the core regions in both LSU rRNA molecules are
comparable, indicating the absence of functional differences between
the C- and D-rRNA molecules. In addition to the structural comparisons,
we addressed the question of functional differences between the C- and
D-rRNA molecules by knocking out the C- and D-rRNA gene units and
analyzing the resulting phenotype. These studies strongly support the
view that the C- and D-rRNA molecules are not functionally different.
The presence of the rRNA of one of the two gene units was sufficient to
allow the parasite to undergo full development within the mosquito and
for the production of infectious sporozoites. Slight growth retardation of the oocysts of both C- and D-ko parasites might indicate that the
existence of two S-type gene units represents a gene dosage effect,
comparable to the existence of multiple rRNA genes or genes encoding
ribosomal proteins in other organisms (43-45). The fact that the
growth retardation of oocysts seems more pronounced when the more
highly transcribed D-unit is disrupted rather then the C-unit supports
this conclusion. Similarly, the pattern of transcription in other
apicomplexan parasites suggests that these parasites maintain the
minimum number of rRNA gene units of equivalent function (7-9). A gene
dosage effect has also been suggested for the expression of two
identical gene copies encoding elongation factor-1a in
Plasmodium (46), and disruption of one of these copies
influenced the growth rate of the
parasites.5
During development in the liver and blood of the host, P. berghei expresses two A-type rRNA gene units, the A- and B-type. All evidence so far indicates that both gene units are identical and
expressed simultaneously, which might imply a comparable gene dosage
effect for the A-type rRNA gene units. So far we were unable to knock
out one of the A-type genes, excluding the collection of supportive
evidence for a gene dosage effect. Comparison of the structure of the
A- and S-type SSU and LSU rRNA molecules demonstrates also that between
the A- and S-type molecules structural evidence is lacking in support
of a distinct classification of A- and S-type ribosomes in P. berghei which might be related to function. In all core
regions, the secondary structures of the A- and S-type
molecules are essentially identical, and the extensive differences are
overwhelmingly confined to the highly variable expansion regions whose
structure is well less defined among eukaryotes. The P. berghei LSU rRNA molecules match the eukaryotic consensus sequence
at all positions known to be associated with catalytic activity. If
sequence differences between the core regions of different rRNA
molecules result in the presence of functionally different ribosome
types in Plasmodium, it might be expected that those
sequence differences would be conserved between different
Plasmodium species or that additional species-specific variation at these positions would be observed. However, all positions reported to be variant between the core regions of the P. falciparum A- and S-type LSU rRNA molecules are invariant in
P. berghei, much more similar to the P. falciparum A-type, and identical to the eukaryotic consensus
sequence. This includes the variant sites in the GTPase center where
the P. berghei A- and S-type molecules are identical
to the P. falciparum A-type LSU sequence. The rate of GTPase
utilization by the ribosomes of Plasmodium was considered as
a possible means to control development in the light of different developmental stages containing either S- or A-type ribosomes (21).
This control mechanism will thus be absent in P. berghei because of the similarity of the GTPase site in all LSU rRNA molecules.
During development of the parasite in the mosquito not only S-type rRNA
molecules but also rRNA molecules derived from the A-type rRNA genes
are present. In situ RNA hybridization revealed that a
significant proportion of the rRNA of ookinetes and young oocysts is
A-type that originates from the fertilized female gamete (14, 19). In
this study we found also that mature oocysts and sporozoites still
contain significant amounts of A-type SSU and LSU rRNA molecules, up to
50% of the total amount. The presence of A-type rRNA in sporozoites
was an unexpected observation because in other studies it had been
reported that A-type rRNA was barely detectable in this stage of
development (15, 19, 47). The presence of both A- and S-type ribosomes
in mosquito stages, such as immature (19) and mature oocysts (this
study), challenges the suggestion that the replacement of different
ribosome types has arisen as an adaptive mechanism for efficient
protein synthesis during growth in the two very different environments
found in the host and the mosquito vector (24). Instead, a mechanism of
augmentation of the ribosome population by the introduction of
additional ribosome forms can be considered. Moreover, it extends the
period during which it might be possible for P. berghei to make use of hybrid ribosomes containing both A- and S-type rRNA molecules (19). Unfortunately we were unable to obtain parasites with
both S-type rRNA gene units disrupted; therefore we were unable to
address the question of whether the transcription of only A-type rRNA
gene units would be sufficient for production of infectious sporozoites.
In conclusion, our results do not support the hypothesis that the
structural differences between the rRNA molecules result in the
presence of functionally different ribosome types. The observations of
the lack of structural differences in core regions of all rRNA
molecules, the lack of temporal differences in expression of the S-type
rRNA gene units, the observation that only one copy of the S-type rRNA
genes is sufficient for complete development, and the fact that both A-
and S-type ribosomes are active in the mosquito all indicate functional
equivalence of the different ribosome types of P. berghei.
These findings do not exclude that the differences observed in rRNA
molecules of other Plasmodium species do have functional
significance. In contrast to P. berghei, the secondary
structures of the P. vivax O-type SSU rRNA genes and the
P. falciparum S-type LSU rRNA genes vary in a manner
atypical of other eukaryotes. However, based on the result of P. berghei one could question whether the presence of functional
distinct ribosomes types is a prerequisite for completion of the
complicated Plasmodium life cycle in two different hosts.
The question remains why Plasmodium parasites have evolved
such an atypical and unique organization of stage-specifically
expressed, unlinked rRNA genes that can produce structurally different
rRNA molecules. A possibility is that the unlinked organization is a
relic of evolution, and the structural differences result from a lack
of regular homogenization as in the tandem array organization of rRNA
genes of other eukaryotes. These differences might then accumulate but
do not significantly alter function. It has been shown that gene
conversion does occur among the different rRNA gene units of
Plasmodium (48), and the evidence reported in P. berghei that one of the S-type SSU rRNA genes is a hybrid between
an A-type SSU rRNA gene and the other S-type SSU rRNA gene (23) might
indicate that significant homogenization can occur among the unlinked
gene copies. Another explanation for the atypical organization might
lie in the regulation of expression of the rRNA genes. By maintaining
unlinked copies under control of different promoters, the parasite may
be able to adjust the production of the rRNA molecules depending on the different needs during different phases of development under different environmental conditions. In the relatively short lived mosquito the
parasite has to multiply rapidly and efficiently for efficient transmission to a new host. This may require switching on the expression of additional rRNA genes, the S-type genes, to be able to
fulfill the need for the production of sufficient numbers of ribosomes
in the rapid growing oocyst stage. The significance of the
stage-specific expression of structurally distinct ribosomes by
P. berghei will be illuminated only by gene modification or promoter replacement experiments.
We thank Hans Kroeze for technical support and
the malaria unit of the Department of Medical Microbiology, Nijmegen,
for the supply and dissection of mosquitos.
*
This research was supported by the Research Council for
Earth and Life Sciences, the Netherlands Organization for Scientific Research, and the INCO-DC program of the European Community Contract CT
960052.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ301624, AJ298079, AJ298080, AJ298081, AJ298082. and
AJ298083.
¶
Present address: Division of Parasitology,
Biomedical Primate Research Center, Rijswijk, The Netherlands.
Published, JBC Papers in Press, April 5, 2001, DOI 10.1074/jbc.M101234200
2
R. M. L. van Spaendonk, A. van Wigcheren, C. J. Janse, and A. P. Waters, unpublished data.
3
R. R. Gutell, S. Subashchandran, M. Schnare, Y. Du, N. Lin, L. Madabusi, K. Muller, N. Pande, N. Yu, Z. Shang, S. Date,
D. Konings, V. Schweiker, B. Weiser, and J. J. Cannone, manuscript in preparation.
4
R. M. L. van Spaendonk, unpublished data.
5
M. A. Sperança, H. A. del Portillo,
C. J. Janse, and A. P. Waters, unpublished observations.
The abbreviations used are:
LSU, large subunit;
SSU, small subunit;
ITS, internal transcribed spacer;
ETS, external
transcribed spacer;
ko, knockout;
PCR, polymerase chain reaction;
kb, kilobase pair(s);
bp, base pair(s);
PCRaf, PCR amplified fragment;
wt, wild-type.
Functional Equivalence of Structurally Distinct Ribosomes in the
Malaria Parasite, Plasmodium berghei*
,,,, and
Department of Parasitology, Leiden
University Medical Centre, Postbus 9600, 2300 RC Leiden, and the
§ Department of Medical Microbiology, Section Parasitology,
University Hospital St. Radboud
Nijmegen 6525 ED, The Netherlands
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
zap-SK (Dr. M. Ponzi,
Instituto Superiore di Sanità, Roma, Italy). This library was
screened with a 653-bp fragment from the B-LSU rRNA gene, derived from
clone pPbL4.1 after digestion with HindIII and
AvaI. The final wash was performed at 60 °C in 1 × SSC, 0.5% SDS. Six clones positive for the 3'-end of the LSU gene and
with different insert sizes were obtained. Comparison of the
restriction digestion patterns of these clones with the known
restriction maps (11) and partial sequence analysis extending into the
already sequenced regions of the LSU rRNA genes showed that two clones contained the 3'-end of A-type LSU rRNA genes, three clones the 3'-end
of the C-, and one clone the 3'-end of the D-LSU rRNA gene. Subclones
made by amplification of the 3'-ends of the A-type, C-, and D-LSU rRNA
genes were sequenced completely.
Oligonucleotides specific for the ETS, SSU, ITS1, ITS2, and LSU regions
of the four rRNA units (A/B/C/D) of P. berghei
View larger version (42K):
[in a new window]
Fig. 1.
Generation of ko parasites containing a
disrupted or replaced C- or D-rRNA gene unit. Panel A,
schematic representation of the rRNA gene units of P. berghei (A/B-, C-, and D-units) and their chromosomal location.
The SSU rRNA gene, the 5.8 S, and the LSU rRNA genes are shown in
addition to the 5'-ETS and the ITS1 and ITS2. Arrows
indicate restrictions fragments that have been cloned (see
"Experimental Procedures") or that are used for identification of
the different units by Southern analysis (see panels E and
F). Fragments that are used as probes for Southern analysis
of genomic DNA of ko parasites are boxed (C/D ETS, C/D-ITS1,
D-ETS). Panel B, vectors used to knock out the C- or D-rRNA
gene unit. To disrupt the wt C- or D-unit parasites were transfected
with vector pMD207, linearized at an unique SpeI site.
Vector pMD207 contains an incomplete copy of the C-SSU rRNA gene as a
target sequence and the pyrimethamine-resistant P. berghei
DHFR/TS gene (PyrR1). To replace the wt C- or D-unit, the replacement
vector 395A or 387A cut with BamHI and EcoRI was
used. Vectors 395A and 387A both contain the pyrimethamine-resistant
Toxoplasma gondii DHFR/TS gene (PyrR2). Vector 395A contains
the complete S-type ITS1 region and a central part of the D-LSU as
target sequences, whereas vector 387A contains the same part of the
D-LSU and a fragment of the D-ETS as target sequences. Panel
C, schematic representation of the disrupted or replaced C- and
D-rRNA gene units. Integration of vector pMD207 results in a disruption
of the rRNA unit by an incomplete and a complete copy of the SSU gene
separated by pUC19 plasmid sequences containing the selectable marker
PyrR1 (Cdis and Ddis). In addition, two EcoRI restriction
sites are introduced by the integrated pMD207 vector (at the 3'-end of
the incomplete copy and at the 5'-end of the complete copy of the SSU
gene). Integration of vector 395A into the wt C- or D-rRNA unit results
in the replacement of the complete 5.8 S gene, the ITS2 region, and the
5'-end of the LSU rRNA gene by pUC19 plasmid sequences containing the
selectable marker PyrR2 (Crep1 and Drep1). Integration of vector 387A
into the wt D-rRNA unit results in the replacement of the 3'-end of the
D-ETS, the SSU gene, the ITS1 region, the 5.8 S gene, the ITS2 region,
and the 5'-end of the LSU gene by pUC19 plasmid sequences containing
the selectable marker PyrR2 (Drep2). Integration of vector 395A as well
as 387A results in the introduction of an HindIII
restriction site at the 3'-end of the ITS1 and a KpnI
restriction site at the 5'-end of the remaining LSU sequences. Arrows indicate restriction fragments that confirmed
correct disruption or replacement of the rRNA units by Southern
analysis (see panels E and F). Panel
D, hybridization of oligonucleotides specific for the different
rRNA gene units to chromosomes that are separated by field inversion
gel electrophoresis. All A/B unit-specific probes hybridized with equal
intensity to chromosome 12 and to 7. Hybridization of pUC19 shows the
integration of the disruption vector in chromosome 5 and 6 in C-ko and
D-ko parasites, respectively. Panel E, the genotype of ko
parasites before mosquito transmission analyzed by Southern blot
hybridizations. To demonstrate correct integration of vector pMD207
into the C-SSU rRNA gene, DNA was digested with EcoRI and
hybridized to the C/D-ETS probe. In wt parasites, a 14-kb fragment of
the C-unit (panel A) and a 44-kb fragment (not shown) of the
D-unit hybridize to this probe. In the ko parasites, the introduction
of an EcoRI restriction site at the 3'-end of the incomplete
copy of the SSU gene results in a 3-kb fragment (panel C) in
Cdis1 and Cdis2 and a 20-kb in the Ddis parasites (not shown) which
hybridizes to the C/D-ETS probe. To show integration of pMD207 in the
D-SSU rRNA gene, DNA was digested with HindIII and
KpnI and hybridized to the C/D-ETS probe. In wt parasites an
18.4-kb HindIII/KpnI fragment (not shown) of the
C-rRNA gene unit and a 5.2-kb KpnI fragment of the D-rRNA
gene unit (Fig. 1A) hybridize. In the ko parasites an
additional HindIII is introduced at the 3'-end of the ITS1,
resulting in a 20.4-kb HindIII in Cdis parasites (not shown)
and a 7.1-kb fragment (Fig. 1C) in Ddis1 and Ddis2. To
demonstrate integration of vector 395A, DNA was digested with
HindIII and EcoRI and hybridized to the C/D-ITS1
probe. In wt parasites, an 8.8-kb HindIII/EcoRI
fragment of the C-rRNA gene unit and a 17.3-kb HindIII
fragment specific for the D-rRNA gene unit (panel A)
hybridize to this probe. In the ko parasites an additional
HindIII site is introduced at the 3'-end of the ITS1
resulting in a 3.5-kb EcoRI/HindIII fragment in
Crep1 and a 12.8-kb HindIII fragment in Drep1 parasites
(panel C). To show integration of vector 387A, DNA was
digested with KpnI and hybridized to the D-ETS probe. In wt
parasites a 5.2-kb KpnI fragment of the D-rRNA gene unit
(panel A) hybridizes, whereas in ko parasites a 7.5-kb
KpnI fragment (panel C) is present. Panel
F, the genotype of ko parasites after mosquito transmission
analyzed by Southern blot hybridizations. As before mosquito
transmission, in Cdis1 and Cdis2 a 3-kb EcoRI fragment
specific for a disrupted C-unit, in Crep1 a 3.5-kb
EcoRI/HindIII fragment specific for a replaced
C-unit, in Drep1 a 12.8-kb HindIII fragment specific for a
replaced D-unit, and in Drep2 a 7.5-kb KpnI fragment
specific for D-unit replacement are visible. However, in addition to
the 7.1-kb KpnI/HindIII fragment specific for a
disrupted D-unit (panel B), in Ddis1 and Ddis2
after mosquito transmission a 5.2-kb KpnI fragment specific
for wt parasites (panel A) is visible as well. These wt
parasites arise as the result of reversion events caused by reversal of
the integration mechanism leading to excision of the insertion vector.
E, EcoRI; K, KpnI;
H, HindIII; S, SpeI; and
B, BamHI).
HDB (26). In both vectors a 723-bp fragment of the D-LSU rRNA gene was cloned downstream of the selection cassette. To obtain this fragment, plasmid pPbL4.8 (see above) containing 1.6 kb of the D-ITS2
and 3.2 kb of the D-LSU rRNA gene was digested with TaqI and
HindIII. After size fractionation and purification from gel, the 723-bp TaqI/HindIII fragment was cloned in
pBS/KS digested with ClaI and HindIII. The
HindIII site was destroyed by religation after filling out
the HindIII-digested clone. The resulting clone was digested
with KpnI and EcoRI, and the insert containing
nucleotides 2569-3292 of the D-LSU rRNA gene was subsequently cloned
into plasmid
pDBDTmHDB digested
with KpnI and EcoRI, giving rise to plasmid
pDBDTmHDB/D-LSU. To
create vector 395A, the ITS1 that is 100% identical between the C- and
the D-unit,4 was PCR
amplified from plasmid pPbSL8.8 (24) with the oligonucleotides L427R
and L412R (Table I; 30 cycles, annealing at 55 °C, 45-s extension),
which introduced unique HindIII sites at either end of the
ITS1. The cloning of this 522-bp amplification product (probe C/D-ITS1,
Fig. 1A) into the unique HindIII site in plasmid pDBDTmHDB/D-LSU
resulted in the formation of plasmid 395A. To create vector 387A,
plasmid pPbS5.2 (23) containing the entire D-SSU rRNA gene flanked by a
2.9-kb upstream sequence and by 0.2 kb of the ITS1 was digested with
NheI and SacII. After size fractionation, an
2048-bp NheI/SacI fragment containing 1480 bp of
the upstream sequence and 568 bp of the D-SSU rRNA gene was purified
from gel and cloned into pBS/KS digested with SpeI and
SacII. From this clone a D-unit-specific 868-bp region of
the D-ETS (probe D-ETS, Fig. 1A), located 612 bp upstream of
the D-SSU rRNA gene, was PCR amplified with the oligonucleotides L372R
and 307A (Table I). After digestion with HindIII, the
amplification product was cloned into
pDBDTmHDB/D-LSU, giving rise to replacement vector 387A.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Size and identities of the SSU, 5.8 S, and LSU genes of the four rRNA
units
Sequence differences in core regions of the LSU rRNA genes between P. berghei, P. falciparum and other eukaryotes
View larger version (56K):
[in a new window]
Fig. 2.
The presence of RNA transcripts of
the different rRNA units in wt and ko parasites determined by Northern
analysis and dot spot experiments. Panel A, the
presence of SSU and LSU transcripts of the different rRNA units in
blood stages, mature oocysts, and sporozoites of wt parasites,
determined by hybridization of RNA to specific oligonucleotides.
Panel B, the large fragment (~3 kb) of the LSU transcript
of the different units in mature oocysts of wt parasites. Small
differences (153-242 bp) exist in the size of the LSU genes between
the A-type and S-type, resulting in the separation of A- and S-type
transcripts. Based on the relative hybridization intensity of these
fragments to oligonucleotides (see panel C) that recognize
all transcripts we estimated by PhosphorImager analysis that the ratio
of A- and S-type transcripts is 60%:40%. Panel C,
hybridization of oligonucleotides specific for the SSU or LSU genes of
the different rRNA units (see Table I) to plasmids containing
restriction fragments of the individual units. These hybridizations
show the specificity of the probes and were used for measurement of the
specific activity of labeled oligonucleotides for the determination of
the relative abundance of C- and D-transcripts in mature oocysts in dot
spot experiments by PhosphorImager analysis (see panel A).
Panel D, the absence of C- and D-LSU transcripts in mature
oocysts of C- and D-ko parasites, respectively, as shown by dot spot
hybridization with oligonucleotides specific for the different units.
Panel E, the presence of LSU and SSU transcripts in blood
stages and mature oocysts of wt and ko parasites. SSU and LSU
transcripts of the A-type units are present in mature oocysts of both
wt and ko parasites; transcripts of the C- and D-gene units are absent
in C- and D-ko parasites, respectively.
View larger version (57K):
[in a new window]
Fig. 3.
Phenotype of wt parasites and of ko parasites
containing a disrupted or replaced C- or D-rRNA gene unit, showing a
small retardation of the growth of oocysts. Panel A,
the development of gametocytes and ookinetes and the numbers of oocysts
of ko parasites are comparable to those of wt parasites. A significant
difference (* = p 
0.05; t test and
Kruskal-Wallis test) is observed in the size of oocysts (mean size with
S.D. is shown) at 10 days after mosquito infection. The conversion rate
of asexual parasites into gametocytes is the percentage of ringforms
that develop into gametocytes. The conversion rate of gametes into
ookinetes is the percentage of female gametocytes that develop in
vitro into ookinetes (28, 30). All ko parasites, except for Ddis1,
produced mature oocysts containing sporozoites at day 10 after
infection. Panel B, the size of oocysts during development
of ko parasites (Ddis1) and wt parasites. Panel C,
10-day-old oocysts of ko and wt parasites, photographed using a light
microscope at a ×400 magnification. Knockout parasites produced normal
numbers of oocysts. The only difference between ko and wt parasites is
a small one, in the size of mature oocysts.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed. Tel.:
31-71-526-5069; Fax: 31-71-526-6907; E-mail: Waters@lumc.nl.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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