volume 12 Number 14 1984
Nucleic Acids Research
Two major sequence dgsses of ribosomal RNA genes in Piasmodium berghei
John B.Dame*, Margery Sullivan1 and Thomas F.McCutchan
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, MD 20205, and k3enex Corporation, Rockville, MD 20850, USA
Received 17 February 1984; Revised and Accepted 19 June 1984
ABSTRACT
Primary sequence differences have been found between two different
ribosomal DNA (rDNA) units of the rodent malaria parasite,
Piasmodium berghei, within the coding areas of both the small and large
ribosomal RNAs (rRNA). The coding regions of rONA unit A are protected from
nuclease SI digestion by rRNA isolated from asexual blood stage parasites.
Under the same conditions of analysis, the comparable coding regions from
unit C are cut into small pieces by nuclease SI, the largest being 1.1 kb.
Analysis of heteroduplexes of the respective DNA clones from units A and C
by electron microscopy reveals that the two units differ in the 5' flanking
and internal transcribed sequences and that there are extensive sequence
differences in the DNA coding for the mature large rRNA. No introns were
detected in either rDNA unit. The data shows that unit A is transcribed in
blood stage parasites and that unit C is not.
INTRODUCTION
The copy number and arrangement of ribosomal genes in P. berghei is not
typical of eukaryotic organisms. Commonly there are many tandemly repeating
ribosomal genes in the DNA of eukaryotes, and the transcription of these
active genes is thought to be constitutive. However, in P. berghei there
are only four genes, and they are not in close proximity to one another (1).
The differences in organization of ribosomal genes in this parasitic
protozoan raises questions about whether or not the transcriptionai control
of these genes is also fundamentally different than that of other
eukaryotes.
From previous studies (1,2,3), it is known that although the four genes
are very similar they fit into two classes based on restriction analysis.
Units A and B have in common short internal transcribed sequences (ITS) and
four specific restriction nuclease cleavage sites within the coding areas.
Units C and D have longer ITS and also lack three of the four restriction
sites in common between units A and B. Here we use SI nuclease analyses and
electron microscopy to compare cloned genes from the two classes. We find
© IRL Press Limited, Oxford, England.
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that 1) the genes differ in the 5' flanking and ITS regions, 2) there are
extensive sequence differences in the DNA coding for the mature large and
small ribosomal RNAs (rRNA), and 3) one of the units is not transcribed in
blood stage parasites.
These data suggest that the genes are evolving in an
independent manner and are potentially under different transcriptional
control.
METHODS
Parasites
The NYU2 strain of P. berghei which no longer produces sexual forms was
syringe-passaged in white mice.
Asexual blood stage parasite were prepared
from infected blood (50-70% parasitemia) by saponin treatment as described
(1).
Clones of P. berghei rDNA
Clones of unit A and C were prepared previously (2) from Eco Rl
fragments of P. berghei DNA in X phage Charon 4A.
Unit A was formerly
referred to as the 7.8 + 6.9 kb unit and unit C as the 14 kb unit (1,2).
Subclones of unit A in pBR322 (1) and subclones of unit C wi11 be referred
to by their length in kb.
Fragments of unit C were subcloned as described
for unit A (1), except pSV7186 (4) was used as the cloning vector.
the two units are compared in Figure 1.
Maps of
All plasmid DNA was isolated as
supercoil DNA (5).
RNA
Total RNA and RNA from purified ribosomes of asexual blood stage
parasites were prepared by phenol-chloroform extraction as described (1).
Nuclease SI Analysis
SI analysis was performed essentially as described (6). Supercoiled
DNA was digested with the appropriate restriction enzymes (see Figure 1) to
release the rDNA insert from the plasmid cloning vector without internal
cleavage.
The digested DNA was phenol-chloroform extracted and ethanol
precipitated.
Two 1 ug samples of each digested DNA dissolved in 10 mM
Tris-HCl, pH 7.5-1 mM EDTA, pH 8.0 were mixed with 100 ug of calf liver tRNA
plus either 20 ug of total RNA or 15 ug of ribosomal RNA and ethanol
precipitated.
Nucleic acid precipitates were washed with 80% ethanol, dried
and resuspended in 30 ul of hybridization buffer containing 40 (DM PIPES,
pH 6.4-1 mM EDTA, pH 8.0-0.4 M NaCl-80% forroamide.
The DNA in the samples
was denatured for 15 roin at 72°C then transferred immediately to 49°C and
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B
Unit A .
ZJ?
E
Unit C
p
H
u
1 Kb
Figure 1. Maps of the rRNA coding regions of rONA units A and C of
P. bergTiei.
Panel A. The position of the small rRNA coding region in unit A was
determined by R loop analysis. In the electron micrograph shown arrows
indicate the origin and terminus of the R loop of the small rRNA formed
between clone 7.8 and total RNA. The mean values (±SD) for the lengths of
the 3 regions defined by the R loop are: 5' flanking region, 2976
(± 217 bp); R loop, 2058 (± 260 bp); 3' flanking region, 2763 (± 176 bp)
(n=3). The measured lengths were adjusted by a conversion factor of 0.9 to
conicide with the length of the molecule as measured by agarose gel
electrophoresis.
Panel B. Bars below the map lines indicate the location and expanse of
the rRNA genes. The approximate positions of the coding regions for the
small, large and 5.8S rRNAs in Unit A and Unit C were determined by
restriction enzyme analysis (1, 3). The position of the small rRNA coding
region in Unit A was determined by R loop analysis (Panel A ) , and the
position of the coding region of the large rRNA in Unit A was from nuclease
SI data (see below). The positions of the two major rRNA coding regions in
Unit C were determined by heteroduplex formation with Unit A (see below).
The positions of the 5.8S RNA coding region 1n the two units (marked with •)
were inferred from the heteroduplex analysis (see below). The arrow
indicates the position of the natural nick site in the large rRNA. E, Eco
Rl; H, Hind III; K, Kpn I; S, small rRNA gene; L, large rRNA gene.
hybridized for 3 h.
Nuclease SI (60 U) was added to each reaction in 300 ul
of ice cold nuclease-Sl buffer and digested at 37°C for 30 m1n.
The
reaction was stopped by the addition of 50 pi of 4 M ammonium acetate-0.1 M
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EDTA, and 2 ul of 10 mg/ml carrier tRNA was added. The reaction mixture was
extracted once with phenol-chloroform and precipitated with 1 volume of
isopropanol. Each sample was dissolved in 10 mM Tris-HCl-1 raM EDTA, pH 8.0
and alkali denatured by the addition of NaOH to 0.05M. One half of each
sample was run on a 2% alkaline agarose gel (6), a Southern blot was
prepared and was hybridized with polynucleotide kinase-labeled total RNA
probe (1).
R Loop and Heteroduplex Analysis
R loops were formed under conditions described for SI nuclease analysis
and spread for electron microscopy as described (7). Heteroduplexes were
formed between plasraid clones from units A and C. Each plasmid was digested
with restriction enzyme(s) to free the cloned insert from the plasraid
vector. Two hundred nanograms of each were mixed together and denatured in
22.5 pi 0.11 N NaOH-22 mM EDTA at 25°C for 15 minutes. The reaction mixture
was neutralized by the addition of 2.5 pi of 2 M Tris HC1 pH 7.2 and made
50% in formamide by adding 25 ul of deionized formamide. The heteroduplex
reaction was left at 25°C overnight and spread for electron microscopy as
described (7).
RESULTS
SI analysis of cloned DMAs reveals primary sequence differences between
unit A and unit C in the coding areas for both the large and small rRNAs.
In these experiments rRNA from asexual blood stage parasites was used to
determine the regions of homology between cloned DNAs and mature RNA. The
DNA clone 5.6 contains the entire small rRNA gene of unit A but none of the
large rRNA gene. This clone yields a single fragment, 2.15 kb in length,
that is protected from SI digestion by total RNA or rRNA (Figure 2). This
corresponds to the size of the mature small rRNA (2). The DNA clone 8.8
contains the entire small rRNA gene and more than 3kb of the large rRNA gene
from unit C. Under the same conditions of analysis this clone yields no
protected fragments larger than 1.1 kb. It is then apparent that the small
rRNA from blood stage parasites does not completely protect the unit C small
rRNA gene and therefore that the two units are not homologous in sequence.
The coding region for the large rRNA was analyzed in a similar manner also
taking into account that this RNA is naturally cleaved j_n vivo 0.8 kb from
the 5' end to yield two pieces (1,2). The DNA clone 7.8 contains the entire
gene for the small rRNA and 2.0 kb of the 5' portion of the gene for the
large rRNA of unit A (1,2). The 2.15 kb fragment coming from the small rRNA
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7.8
5.6
8.8
3.3
kb
•
A A
0.8_
•
-3-1
-2.3
-08
Figure 2. Nuclease SI analysis of units A and C hybridized to total and
ribosomal RNA.
Plasmid subclones 7.8, 5.6, 8.8, and 3.3 were analyzed using SI
nuclease as described in Methods. In the autoradiogram shown, the sample in
the first lane in each pair was hybridized prior to nuclease SI digestion
with total RNA, the sample in the second lane with ribosomal RNA. Where
samples of clones 5.6, 3.3 and 8.8 were treated without the inclusion of
homologous RNA, no rDNA fragments resistant to SI nuclease were detected
(not shown).
gene and two fragments from the large rRNA gene, 1.2 and 0.8 kb, were
detected. These results are expected since the rRNA used for RNA-DNA duplex
formation is naturally cleaved. Analysis of DNA clone 3.3 which extends
1.1 kb more into the large rRNA gene of unit A than clone 7.8 also yields
the 0.8 kb fragment. This confirms that the 0.8 kb fragment represents the
region of the large RNA gene from its 5' end to the point of its natural
cleavage site as shown previously (2). The same experiment also yields a
2.3 kb fragment showing that the unit A DNA is homologous with mature RNA at
least to the end of this fragment. Total RNA, which contains some unnicked
rRNA from the large ribosomal genes, protects a 3.1 kb fragment in addition
to the 2.3 and 0.8 kb fragments. Clone 8.8 which contains the coding
regions from unit C that are equivalent to the sum of clones 3.3 and 5.6
from unit A is not protected by RNA in a similar manner. No fragment larger
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7.8
3035
±346
2075
-.362
1004
+ 112
8.8
Figure 3. Heteroduplex of 7.8 x 8.8.
Panels A and B. Arrows point to important features indicating the
origins and termini of major double stranded regions, a short
double-stranded region, and small regions of non-homology interrupting the
large rRNA coding region. Panel C. A schematic drawing of a typical
heteroduplex is presented which was generated from the measurement of 15
molecules. The average length of each feature with the standard deviation
is given in bases. The measured lengths were adjusted by a conversion
factor of 0.80 to coincide with the length of each molecule as measured by
agarose gel electrophoresis.
than 1.1 kb is detected indicating that the mature rRNA from asexual blood
stages is not completely homologous to unit C. Thus, units A and C differ
in both large and small rRNA coding regions, and unit A is the template of
the predominant transcript in the asexual blood stages.
In order to determine the nature and extent of sequence differences
between units A and C, heteroduplexes of the respective ONA clones were
analyzed by electron microscopy. DNA inserts were excised from supercoiled
plasmids and the appropriate pairs were mixed [7.8 (A) vs 8.8 (C) and 3.3
(A) vs 8.8 (C)] denatured, reannealed, and spread under the conditions
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described in the legend of Figure 3. Analysis of the 7.8 (A) vs 8.8 (C)
heteroduplexes is shown in Figure 3B. The region tested for homology
includes DMA that is 51 to both units, the entire small rRNA genes, the ITS,
and 2 kb into the 5' end of the large rRNA gene. The result indicates that
there is no homology up to a point 3.0 kb from the 5' end of clone 7.8 (A)
and 1.0 kb from the 5' end of clone 8.8 (C). At this point a duplex forms
and extends for 2.1 kb, which is the small RNA gene. (This has been
confirmed by restriction nuclease and R loop analyses; Fig. 1). Regions of
non-homology predicted by SI analysis between units A and C in the small
rRNA gene are apparently too small to be visualized by electron microscopy.
Between this duplex and the start of the large rRNA gene the only region of
homology is a 160 base pair duplex which likely corresponds to the 5.8S RNA
gene; the remainder of the ITS did not hybridize. The location of the 5.8S
gene is consistent with restriction analysis (3). Two regions of
non-homology interrupt the heteroduplex between units A and C in the coding
area for the large rRNA. These regions are approximately 150 bases in
length and occur at 0.5 and 0.8 kb from the 5' end of the large rRNA gene.
The length of single stranded DNA in both clones is nearly identical
suggesting that sequence differences rather than introns are responsible.
To test homology nearer the 3' end of the large rRNA gene, we tested clones
3.3 (A) and 8.8 (C). This extends the comparison of the two units by 1.1 kb
in a 3' direction from the above analysis. The study reveals three
additional regions of non-homology (Figure 4). The two previously noted
non-homologous segments 0.5 and 0.8 kb from the 5' end of the large rRNA
coding region seen in Figure 3 are present in this heteroduplex together
with some non-homologous segments of similar size at about 1.9, 2.3 and 3 kb
from the 5' end.
DISCUSSION
From the results presented it is clear that the unit A gene is a
template for rRNA transcribed in blood stage parasites, whereas unit C does
not appear to be transcribed since it has numerous regions of non-homology
with the mature rRNA. During the preparation of this manuscript other
investigates studying the rONA of P. lophurae (8,9) and P. falciparum (10)
have reported finding introns in the rRNA genes studied. Our results differ
in that no interruptions in the coding regions from the small or the large
rRNA were found in unit A and the interruptions in unit C appear to be due
to sequence divergence rather than to the presence of Introns. In light of
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33
288
*356
8.8
Figure 4.
Heteroduplex of 3.3 x 8.8.
Panel A. Arrows point to important features indicating the origin of
the double strand region and the five regions of non-homology within the
large rRNA coding region. In Panel B is presented a schematic drawing of a
typical heteroduplex generated from the measurement of 19 molecules. The
measured lengths were adjusted by a conversion factor of 0.88 to coincide
with the length of each molecule as measured by agarose gel electrophoresis.
our results, it is possible that the P. lophurae rDNA unit examined (9) may
not be the gene active in rRNA transcription 1n this organism, since we have
shown that an rDNA unit without introns is present in P. berghei. Further,
it is not clear that an intron has been found in P. falciparum (10), since
no rRNA coding area 3' to the proposed intron has been mapped. If the
P. falciparuai gene pPFrib2 is not fully homologous to the large rRNA in
regions near the 3' end as found in unit C discussed here, the lower than
expected amount of homology to the adjoining region is easily explained
without requiring the presence of an intron.
By comparing the structure of units A and C we may gain some indication
of the origin and possible function of unit C. Unit C may have been derived
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from unit A through duplication and translocation (11), later diverging
either randomly or because of selective pressure. Differences in regions 5'
to each gene are consistant with duplication transposition. Extensive
homology between the coding areas of both units also indicates a common
derivation. The contrast between homology in the exact regions coding for
mature rRNAs and the lack of homology in other areas suggests that the
sequence drift has been influenced by selection. Further there appear to be
localized areas of change in the large rRNA gene that can not be accounted
for either by insertion sequences or by random drift. It may be that
differences in these particular areas reflect a need for localized
functional alterations such as the binding of a different ribosomal protein.
Certainly there are different 5S genes expressed in Xenopus oocytes and
somatic cells (12) and so it may be that unit C is expressed at a different
stage of the parasite life cycle. Alternatively unit C and unit A may not
have a common progenitor and one gene may have integrated into the
Plasmodium genome from a mitochondrial, host or viral source. Mitochondrial
and viral ONA segments are commonly found in genomic DNA (13,14,15). The
similarities among the four units, however, argues against this. For
example there is a common Hind III restriction site that splits the large
rRNA gene of each of the four rDNA units 0.8 kb from the 3' end of the gene
(1). Other arguments against a mitochondrial origin include the presence of
a 5.8S gene which does not occur in mitochondrial rRNA genes (16). It seems
most likely that unit C was derived from a unit A-like progenitor and has
changed under selective pressure. Since the homology between the genes is
exactly in the region producing mature rRNAs, it would also seem likely that
selection was acting on the RNA level and that transcription of the unit C
gene occurs at some stage of the life cycle.
There is a reason to extend this study to the rRNA of less accessible
stages of the parasite. What has been observed could represent a unique
parasite control mechanism that has not been found in other non-parasitic
organisms. Transcription of a new set of rRNAs could be a major step in the
commitment of the parasite to a change in life cycle stage by influencing
the population of messenger RNAs that are translated. Alternatively two
sets of rRNAs could reflect the two host conditions under which Plasmodiuro
lives or simply that different segments of the genome are accessible to
transcriptional apparatus during different stages of the life cycle. Any of
these mechanisms would be of great interest in understanding the parasitic
life style at a genoraic level.
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*To whom inquiries should be addressed
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