1. RNA (1996), 2:134-145. Cambridge University Press. Printed in the USA
Copyright @ 1996 RNA Society.
M. JOHN ROGERS,1 ROBIN R. GUTELL,2,3 SIMON H. DAMBERGER,2 JUN LI,1 GLENN A. McCONKEY,1
ANDREW P. WATERS,4 and THOMAS F. McCUTCHAN1
1Growth and Development Section, Laboratory of Parasitic Diseases. National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20892-0425, USA
2 Department of MCD Biology. Campus Box 347. University of Colorado. Boulder, Colorado 80309-0347. USA
3 Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, Colorado 80309-0215, USA
4 Laboratory of Parasitology, University of Leiden, Leiden, The Netherlands
ABSTRACT
The developmentally regulated transcription of at least two distinct sets of nuclear-encoded ribosomal RNAs
is detected in Plasmodium species. The identification of functional differences between the two sets of rRNAs
is of interest. To facilitate the search for such differences, we have identified the 5.85 and 285 rRNAs from plas-
modium falciparum that are expressed in the sporozoite stage (5 gene) of the parasites' life cycle in the mos-
quito host and compare them to transcripts expressed in the red blood cells (A gene) of the vertebrate host.
This completes the first set of A- and 5-type nuclear-encoded rRNA genes for a Plasmodium species. Analysis
of the predicted secondary structures of the two units reveals the majority of differences between the A- and
5-type genes occur in regions previously known to be variable. However, the predicted secondary structure
of both 285 rRNAs indicates 11 positions within conserved areas that are not typical of eucaryotic rRNAs. Al-
though the A-type gene resembles almost all eucaryotes, being atypical in only 4 of the 11 positions, the 5 gene
is variant in 8 of the 11 positions. In three of these positions, the 5-type gene resembles the consensus nucle-
otides for the 235 rRNA from Eubacteria and/or Archaea. A few differences occur in regions associated with
ribosome function, in particular the GTPase site where the 5-type differs in a base pair and loop from all known
sequences. Further, the identification of compensatory changes at conserved points of interactions between
the 5.85-285 rRNAs indicates that transcripts from A- and 5-units should not be interchangeable.
Keywords: malaria; mosquito stage; ribosome; RNA secondary structure
Plasmodiumfalciparumis unusual in having rRNA gene
setsthat arepresentin low copy number and dispersed
on different chromosomes(McCutchan, 1986;Wellems
et al., 1987;Li et al., 1994a).Unlike most eucaryotes,
two distinct types of cytoplasmicPlasmodium185rRNA
genes have been shown to be developmentally reg-
ulated, one type is predominantly expressed in the
mosquito host and the other in the mammalian host
(Gunderson et al., 1987;Li et al., 1994b).Although the
ratio of the two types of transcripts changes dramati-
cally during the developmental cycleof the parasite, it
is likely that neither transcript disappears entirely at
any point in the life cycle. Certainly there areextended
periods of time when transcripts from both gene sets
are present simultaneously (Gunderson et al., 1987;
Waters et al., 1989).The sequence of two 185 rRNA
INTRODUCTION
AssociationbetweentherRNAs islikely to play anactive
role in the assembly and interaction of the ribosomal
subunits (reviewed in Noller, 1991;Mitchell et al., 1992;
Holmberg et al., 1994),and influences catalysisand ac-
curacy in protein synthesis (reviewed in Noller et al.,
1990,1992).A typicaleucaryotemaintainshomogeneous
copiesof the 185-5.85-285rRNA genesetsencoded in
the nucleus in the form of tandemly repeated clusters
(Long & Dawid, 1980).The human malaria parasite
--
Reprint requests to: T.F. McCutchan, Growth and Development
Section, Laboratory of Parasitic Diseases, Building 4, Room Bl-28,
National Institute of Allergy and Infectious Diseases, National Insti-
tutes of Health, Bethesda, Maryland 20892-0425, USA; e-mail:
mcutchan@helix.nih.gov.
134

2. Structurally different rRNAs in Plasmodium 135
geneshasbeen reported for P.falciparum(McCutchan
et al., 1988).Here we describethe rRNA variation be-
tween thesetwo stage-specifictypes of ribosomes asit
relates to the 5.85 and 285 rRNAs.
The sequenceof a 5.85 rRNA-intemal transcribed
spacer2 (IT52)-285 rRNA gene set from P.falciparum
has been reported, and expression of this gene set in
the blood stagehasbeen demonstrated (Waters et al.,
1995).Following convention (Li et al., 1994a;McCut-
chan et al., 1995),this set is termed the asexual type
(A-type). The question arises as to the nature of the
5.85-285rRNA genesetthat is expressedin the sporo-
zoite stagein the mosquito host (5-type). We describe
here the isolation of a distinct 5.85-IT52-285 rRNA
genesetfrom P.falciparumand demonstrate its expres-
sion in the sporozoite stage. A detailed secondary
structure analysis shows that the 5-type 5.85 and 285
rRNA gene set contains regions of both high conser-
vation and regionswith ahigh degreeof variation with
the A-type gene set, and suggestspossible functional
differences, inferring that genetic exchange between
the two types of rRNA units is restricted.
The 5.85 rRNA genedescribedhere (Fig. 1)is almost
identical to the clone 119described previously as one
of four P.falciparum5.85 rRNA genes(5hippen-Lentz
et al., 1990),with only a single A-+ C change at posi-
tion 64. The 119clone was not found to be expressed
in the asexualstage(5hippen-Lentz et al., 1990).This
5.85 rRNA gene shows only 80% homology with the
asexually expressed5.85 rRNA genesdescribedprevi-
ously (5hippen-Lentz et al., 1990;Waters et al., 1995).
The corresponding P.falciparum5.85 rRNAs can then
be clearly distinguished both by sequenceand size(168
nt for the genein this study compared with 157-159nt
for the asexually expressedgenes).The IT52 (Fig. 1)is
259 bp, compared with 197 bp for the asexually ex-
pressed gene set. Apart from sharing an extreme AIT
bias (82%), the two IT52 sequences share little se-
quencehomology .The 5 nt at the 3' end of the IT52 are
conserved between both P. falciparum gene sets and
three yeast species(van der 5ande et al., 19~2).
The 5' and 3' ends of the 285 rRNA encoded by this
gene set is inferred from secondary structure, phylo-
genetic comparison, and sequenceconservation with
the asexually expressedgene set (Waters et al., 1995).
This correspondsto a4.17-kb285 rRNA gene product,
compared with 3.785-kb 285 rRNA for the asexually
expressedgene. The A and 5 genesare about 80%ho-
mologous, although, as discussed below, the differ-
encesarelocated primarily in variable regions that are
eucaryote specific. Therefore, the 285 rRNAs of P.fal-
ciparumare distinct in both length and sequence.
RESULTS
The sequem~e of a distinct 5.8S-ITS2-28S gem~ set
An rDNA clone was identified by sequenceanalysis
that had only 80% homology with the asexually ex-
pressed285rRNA gene(Waterset al., 1995).It was iso-
lated by hybridization of labeled rRNA to a genomic
library of mung bean nuclease-cleaved P. falciparum
DNA (McCutchan et al., 1984;seethe Materials and
methods). The sequence of this clone revealed that
about 830nt of the 5' end and about 500nt of the 3' end
of the corresponding 285 rRNA gene were missing
(Fig. 1). Thecomplete 5.85-IT52-285genesetwas then
assembledfrom overlapping fragments obtained from
PCRwith genomic DNA astemplate. The primers for
PCRwere designedwith oligonucleotideshomologous
to the 5' end of the P. falciparum 5.85 rRNA genes
(5hippen-Lentz et al., 1990),and to a specificsite in the
5' end of the newly identified 285 rRNA gene (Fig. 1;
seethe Materials and methods). The 3' end of the 285
rRNA gene was amplified with a primer homologous
to a region specific to the newly identified 285 rRNA
gene and one complementary to the conserved 3' end
of the 285rRNA genes.The identity of sequenceover-
lap between the PCRfragments and the genomic DNA
fragment ensured assemblyof the complete DNA se-
quence. The sequenceobtained overlapped with the
185rRNA-IT51-5.85 rRNA 5-type units from p; falcipa-
rum (Rogerset al., 1995),and probably corresponds to
the pPfribl clone of P.falciparumrDNA identified pre-
viously but not characterizedin detail (Langsley et al.,
1983).
Expression of the gene set in the sporozoite stage
Wedetermined the pattern of expressionof this distinct
5.85-IT52-285rRNA gene setduring the life cycleof P.
falciparum.By analogy with the expression of the 185
rRNA genes of P.falciparum(McCutchan et al., 1988)
and other Plasmodiumspecies (Li et al., 1994a),this
genesetwas suspectedto be expressedin the mosquito
host upon differentiation of the parasite into the
sporozoite stage. Therefore, RNA was isolated from
four sources; purified sporozoites from Anopheles
stephensiinfected with P. falciparum, uninfected A.
stephensiasa negative control, ablood-stage culture of
P.falciparum(Trager& Jensen,1976),and RNA from an
uninfected blood culture as a control.
The RNAs described above were used as template
for RT/PCR with the complementary strand synthe-
sized with primer 884(specificto the geneidentified in
this study) or 940, specific to the asexually expressed
gene(Fig. 1). The PCRwas then continued with 883as
the secondprimer, conserved at the 5' end of both 285
rRNA genes.Theproducts of amplification were identi-
fied by hybridization using oligonucleotides comple-
mentary to specific regions of the P. falciparum 285
rRNA genes (Fig. 2; seethe Materials and methods).
The asexually expressed gene hybridized to oligonu-

4. 137Structurally different rRNAs in Plasmodium
RNA source RNA source
0
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o .~
~ &
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o o
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~
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-
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+0--+
940 (A gene) 940 (A gene)884 (S gene) 884(S gene)
cleotide948corresponding to aband of 580bp, whereas
the genein this study hybridized to oligonucleotide 949
as a band of 620bp (seethe Materials and methods).
As expected, RT/PCRusing blood-stage RNA astem-
plate confirmed the presence of transcripts from the
asexually expressed285 rRNA gene described previ-
ously (Waters et al., 1995), with the same gene ex-
pressed at a much lower level in RNA isolated from
purified sporozoites (Fig. 2). By RT/PCRfrom sporo-
zoite RNA, aband of 620bp was obtained that hybrid-
ized to the probe (949;Fig.2)specificfor the new rRNA,
but did not hybridize to aprobe for the previously de-
scribedA-type unit (probe 948).No PCR-derivedband
couldbedetectedusingRNA from uninfectedmosquitos
or with RNA from either infected blood stage culture
or uninfected blood (Fig. 2). No bands were obtained
by PCR from the RNA preparation without reverse
transcriptase, demonstrating that the RNA is not con-
taminated with genomic DNA (data not shown). This
study determines that the new genesetfrom P.falcipa-
rum is expressedin the sporozoite stage, and by con-
vention it is therefore denoted as5-type. The low level
of expression in the sporozoite stage of the asexually
expressed285 rRNA gene is consistent with previous
data regarding the expression of the 185 rRNA genes
of Plasmodium(Gunderson et al., 1987).
Association between the Plasmodium
5.85 and 285 rRNAs sets
The 5.85 rRNA has acommon evolutionary origin with
the 5' end of the 235 rRNA of eubacteria(Nazar, 1980;
Jacq,1981),and is known to basepair with the 5' end
of the 285 rRNA (Paceet al., 1977;Noller et al., 1981).
The presence of distinct p, falciparum5.85 rRNA and
285 rRNA genes of the A-type and 5-type suggests
possible unique interactions between the correspond-
ing gene products of each type that, by analogy with
other eucaryotic systems (Holmberg et al., 1994),may
hinder associationbetween rRNA subunits of the other
type. A secondary structure prediction of the A-type
5.85 rRNA with the 5' end of the A-type 285rRNA and
comparison with that predicted for the 5-type rRNAs
shows this to be the case(Fig. 3). In the base paired
stems that form between the 5.85 rRNA and the 285
rRNA, there are substitutions that convert C-G base
pairs in the A-type to U-A basepairs in the 5-type, and
other changesto or from wobble basepairs (shadedre-
0-
-g,
"'
~
FIGURE 2. Expression of the 5-type 5.85-285 rRNA gene set in RNA isolated from purified p, falciparum sporozoites. Auto-
radiogram of the RT/rCR products with RNA isolated from different sources as template, detected by hybridization to
duplicate filters with r2r]-labeled oligonucleotides 949 (5-specific) and 948 (A-specific). The first strand synthesis with RT
was carried out with either oligonucleotide 940 or 884, as described in the text. Arrow indicates the position of the 600-bp
DNA marker, with the RNA source indicated above each lane.

6. Structurally different rRNAs in Plasmodium 139
TABLE 1. Differences between the P. falciparum A and S 5.85 and 285
rRNA genes.
5.85 and 5'-half
of 285
3'-half
of 285 Total
236
115
52
60
41
19
3
76
36
121
49
51
15
6
127
69
38
31
25
6
0
49
20
58
27
22
4
5
109
46
14
29
16
13
3
27
16
63
22
29
11
1
-
Total number of differences
Differences in base paired stems:
Compensatory changes
Non-compensatory changes
1. Wobble pair
2. Mismatched pair
Insertions or deletions
Transitions
Transversions
Differences in loop regions:
Transitions
Transversions
Insertions
Deletions
cussedbelow, the 5 geneis different at the GTPasesite
(positions 1059:1079and 1084;Table2) and suggestsa
functional difference in the 5-type 285 rRNA. The
other substitutions occur at positions where functional
information is not known, so the significance of these
differencesto the function of the 5-type ribosomeis not
understood. Interestingly, at a few positions (1346;
1600;2271),the 5 gene is more similar to the Eubacte-
ria and/or Archaeaconsensus(Table2). The 5-type 185
rDNA units are usually more variable when compar-
ing related Plasmodiumspecies(Rogers et al., 1995;J.
Li, unpubl. results), perhaps reflecting a higher muta-
tion frequency in 5-type rDNA units. Information on
5-type 285 rRNAs from other Plasmodiumspecieswill
determine whether changes at these positions are a
general feature of the genus.
The secondary structure of the 5-type and A-type
285 rRNAs alsoprovides information for the likely cat-
alytic activity of the two types of ribosomes. The
GTPaseactivity inherent in the ribosome is located in
a defined region in the 5' portion of the 285 rRNA, and
the corresponding part in eubacterial235 rRNA is also
the siteof interaction of anumber of ribosomal proteins
and thiocillin antibiotics (Cundliffe, 1990;Douthwaite
et al., 1993).Interestingly, there is acompensatorybase
pair change and achange in the loop joining two heli-
ces of this region in the 285 rRNA of the A-type and
5-type (Fig. 5). The binding of ribosomal protein L11,
the antibiotic thiostrepton, and the dependence of
magnesium and ammonium ion on tertiary interactions
have been determined from mutants in this domain
Comparison of the A- and 5-type geneswith regions
of the 5.85/285 rRNAs that are usually highly con-
served in Eucaryais informative becauseit shows that
the 5-type 285 rRNA is unusual at a number of posi-
tions {Table2). Of the 11differences between A and 5
genes at positions that are highly conserved in Eu-
carya, the 5 gene is unusual at 8 positions {Table2). In
contrast, the A gene 285 rRNA is uncommon at 4 of
these16positions {positions 338;1426;2271;2476in E.
colinumbering), whereas the A and 5 genes are both
unique at only one position {position 2476). As dis-
TABLE 2. Comparison of the P. falciparum A and 5 285 rRNA genes at sites that are usually highly conserved
Position
(E. coli numbering)
~
Status in Eucarya Comments
338
856
1059
1079
1084
1346
P. falciparum
A gene Sge
A G
C A
G A
C U
A U
G A
G ~ 85%
Only Eucarya with an A; C ~ 58%
Only Eucarya with an A; G ~ 98%
Only Eucarya with a U; C ~ 98%
Only Eucarya with a U; A ~ 99%
Only Eucarya with an Ac; G ~ 99%
856:921 base pair
1059:1079 base pair
1059:1079 base pair
1346'1600basepair
G Rarely a U; G := 75%
c u Only Eucarya with a Uc; C = 99% 1346:1600 base pair
2271 u A Only Eucarya with a U; A =0078%
Status in Eubacterial, Archaea,
and chloroplast phylogenetic
domains;" exceptional
frequencies noted in bold
G in >95%
A few A's
G ~ 99%, no A'sb
C ~ 97%, no U'sb
A ~ 99%, no U's
Eub A ~ 42%, G = 55%;
Arch G ~ 100%
Eub G ~ 93%, a few U's;
Arch G = 100%
Eub U ~ 42%, C = 55%;
Arch C = 100%
Eub G ~ 96%, no U's;
Arch A ~ 65%, no U's
Eub U ~ 99%, no A's;
Arch G ~ 77%, no A's
Eub A ~ 100%, no Y's;
Arch A ~ 70%, C ~ 30%
2419 G A Only Eucarya with an A; G ==99% 2397:2419 base pair
2476 c u Only occurrence of C or U; A ==90%
aY, pyrimidine; R, purine; Eub, eubacteria; Arch, archaea.
bA few chloroplast have a 1059:1079 A:U base pair.
cExcept Aedes albopictus (mosquito) 1346:1600 A:U base pair
ne

9. M.J. Rogers et al.142
A
t
C UUA
AUt C-G AGU GU
,AA UCG G GU A
I. I I I I I I
UGGUAGCA AC U CA CA
...G-C A
G G-C
A-U
U-A
G-C
FIGURE 5. The GTPase site of P. falciparum 285 rRNA. Secondary
structures of the GTPase domain in the P. falciparum 285 rRNA de-
rived from the 5-type and A-type 285 rRNA genes. Secondary struc-
ture of the GTPase site derived from the 5-type 285 rRNA gene is
shown, and differences with the A-type are indicated by arrows. The
sequence shown corresponds to nt 1618-1674 in the 5-type 285 rRNA
and 1354-1410 in the A-type 285 rRNA.
AGUC
A
G
GUA
from E. coli235 rRNA (Ryan et al., 1991;Lu & Draper,
1994). The differences between the A- and 5-type
GTPasedomains (Fig. 5) correspond to basepair 1059-
1079and 1084in E. coli. The A-type rRNA resembles
=98% of eucaryotes, Eubacteria, and Archaea with a
G-C basepair, whereas the 5-type genehasan A-U in
this position, which, to date, is only seenin two chlo-
roplast 235-like rRNAs (Gutell et al., 1993;5.H. Dam-
berger & R.R. Gutell, unpubl. alignments). Also, the
A-type rRNA has an A at the position corresponding
to 1084that is seen in almost all eucaryotes and in
nearly all Eubacteria,Archaea,and chloroplast 235-like
rRNAs (Table2). The 5-type 285 rRNA is unique with
a U at this position. Interestingly, the GI059-CI079:
C1059-G1079mutation in the E. coli GTPasedomain
reducesthebinding of Lll and thiostrepton (Ryanetal.,
1991).Mutation ofAI084:U, which will mimic the 5-type
rRNA (Fig.5),alsoreducesLll andthiostrepton binding
(Ryan et al., 1991)and reduces the NH4+-dependent
tmin the E. coli GTPasedomain (Lu & Draper, 1994).
There may then, asdiscussedbelow, be functional dif-
ferencesrelated to GTPaseactivity between the A-type
and 5-type 285 rRNAs. In contrast, the domain impli-
catedin peptidyl transferaseactivity (Fig. 1)isconserved
between the A-type and 5-type 285 rRNAs (Fig. 4B)
and therefore any differences in tRNA selection and
translationalratesbetweenthe two moleculesarelikely
to be aconsequenceof differencesoutside this domain.
changesthe characterof the preexisting ribosome. Plas-
modiumspeciesmaintain 185-5.85-285rRNAs with dif-
ferent primary sequences differentially expressed
during development. Maintenance of variation within
the central catalyticcomponent of the ribosome (Noller
et al., 1992)of an organism would then seemto relate
to function, although it is difficult to imagine the ben-
efits to an organism of altering any of the catalytic ac-
tivities known to be associatedwith rRNA. At leasttwo
possibilities have precedence: (1)The rate of GTP hy-
drolysis, including that associatedwith the ribosome,
can playa central role in the control of growth and de-
velopment. This ranges from the response to amino
acid starvation in bacteria, ribosome idling, and the
production of "magic spot" in bacteriaby the stringent
response (Cashel & Rudd, 1987),to the association of
GTP hydrolysis associatedwith the rasgene product
and the transformation and signaling in mammalian
cells(Neer, 1995).(2)mRNA:ribosome associationmay
favor translation of particular subsetsof mRNAs (Chen
et al., 1993) and potentially foster developmental
switches.
Analysis of the secondary structures derived from
the 5.85/285rRNAs from the A-type and 5-type genes
shows that the majority of differences occur in regions
of the rRNA where the consequencesof thesechanges
are unknown. However, a few A- and 5-type differ-
encesarein conservedregions of the rRNA that may be
linked to important functions during protein synthesis.
We suggestcharacteristicdifferencesin GTPaseactivity
between the two types of ribosomes. Other evidence,
discussed below, may also support the idea that ge-
netic exchangebetween the types of units is restricted.
Variations on the conserved core of the GTPasesite
have been well studied for the E. coli GTPasedomain
(Ryan et al., 1991).Although not all of the correspond-
ing differences between the A- and 5-type 285 rRNAs
in the GTPasedomains have been characterizedin the
bacterial system, perhaps the significance of these
changesis that they identify a possible functional dif-
ference in the two-ribosome system of P. falciparum
that can be quantified by the methods of Draper et al.
(1993).In Plasmodium,the role of GTP hydrolysis in
protein synthesis may reflect a difference in synthetic
needsin the blood stageto the developing oocyst stage
versusthe relatively quiescentsporozoite.It alsoappears
that interaction with the GTPasesite of the ribosome is
abiologically tolerated target for modulation of protein
synthesis in response to slower rate of growth. Thus,
these differences in the A and 5 genes in this region
of the rRNA may be functionally significant, although
the full significance of the sequenceheterogeneities in
the A- and 5-type genesawaits experimental evidence.
Points of contactand interaction of rRNAs within the
ribosome are central to the assembly and function of
the ribosome. We have determined the expression of
two distinct 5.85 rRNA-285 rRNA gene sets in P.fal-
DISCUSSION
Members of the Plasmodiumgenus have ribosomes
whose rRNA content is developmentally regulated. Al-
though someother organisms maintain heterogeneous
populations of ribosomes (Etter et al., 1994),variation
in thesecasesis probably basedon the replacement or
modification of apreexisting ribosomalcomponent that

10. ""-
Structurally different rRNAs in Plasmodium 143
chain termination method with oligonucleotides assequenc-
ing primers spaced approximately at 300-nt intervals. Se-
quence alignments and other manipulations were with the
Lasergene software (DNASTAR) or Genetics Computer
Group (GCG) sequenceanalysis software package. Nucleo-
tide sequencesreported in this paper have been reported to
GenBank under the accessionnumber 048228.
ciparum,and, by comparing the secondarystructure of
each,we find predictable differences in regions of the
rRNA associatedwith ribosome assemblythrough di-
rect interaction. Compensatory changes associated
with changes involved in Watson-Crick interactions
between the 5.85 rRNA and the 285 rRNA leads one
to believe that a positive advantageexistsin maintain-
ing structure and differences in these structures pre-
vents total homogenization by genetic exchange.
Interactions between the rRNAs of different units ap-
pearsto favor self-association(Fig. 4A,B). This is most
likely to have the effectof limiting geneticexchangebe-
tween units; becauserRNA processing and assembly
into the ribosome is most likely coordinated, biologi-
cal mixing at the RNA level is unlikely. Interactions in-
volved in 5.85-285rRNA basepaired stemsareproven
phylogenetically and experimentally to be points of as-
sociation (Pace et al., 1977; Gutell et al., 1994).The
compensatory changes may reflect the necessity to
maintain the structure of the RNA with regard to its
role in protein synthesis while preserving a two-
ribosome system in a background where genetic re-
combination is occurring (Enea & Corredor, 1991).
Expression of the 5.8S-28S rRNA
Total RNA was isolated as described (Li et al., 1993) from
about 0.5 mL of a blood stage culture of P. falciparum strain
307 (Trager & Jensen, 1976) at approximately 5% parasitemia,
and from about 464,000 purified sporozoites isolated from the
salivary glands of A. stephensiinfected with P. falciparum strain
307 (Li et al., 1993; gift of Dr. R.A. Wirtz). RNA was also iso-
lated from 0.5 mL of uninfected blood, and from two un-
infected A. stephensi. The RNA from each preparation was
resuspended in a total of 50 p,Lof RNase-free water and 10 p,L
taken for analysis by RT/PCR with the Superscript Preampli-
fication kit (GIBCO/BRL), with ONase I digestion and reverse
transcription as described by the manufacturer. The reaction
from the ONase I digestion was divided in two for reverse
transcription with either oligonucleotide 884 (5'-CCCCCCTT
AGTCCTGTG-3') or 940 (5'-CCCACATTAGTGCGGGG-3')
as primer for first strand synthesis. Reactions were then pro-
cessed by PCR as described above, with oligonucleotide 883
(5'-GGCAAATCCGCCGAATTT-3') added to both reactions.
Products from the RT/PCR reaction were then analyzed by
electrophoresis on a 1.5% agarose gel, transferred and hy-
bridized to duplicate filters as described (Rogers et al., 1995),
and screened by hybridization to [32p]-labeled oligonucleo-
tides 948 (5'-CGGTTAATCCTTCGTTTGG-3') and 949 (5'-GG
A GATAATTCTATA TCGTA G-3') .
MATERIALS AND METHODS
Cloning and sequencing of the
5.85-285 rRNA gl~ne set
Secondary structure analysis of the rRNAs
The secondarystructure analysisof the P.falciparum5.85 and
285 A- and 5-type rRNAs was inferred from comparative se-
quence analysis. These secondary structures are available
in postscript format from the World Wide Web site for RNA
secondary structures (Gutell, 1994);the URL for this site is
http:/ /pundit.colorado.edu:8080/root.html. The secondary
structures arebasedon the paradigm that different RNA se-
quences from the same RNA family (e.g., 285 rRNAs) will
fold into a similar three-dimensional structure (Woeseet al.,
1983).Refinement of the secondary structure models for the
235 and 235-like rRNAs is from the availability of nearly 300
sequences(Gutell et al., 1993).The P.falciparumsequences
were aligned with alarge collection of previously aligned Eu-
carya 235-like rRNA sequences,maintaining maximum pri-
mary and secondary structure similarity (Woeseet al., 1983;
Gutell et al., 1985,1994).The computer editor AE2 facilitated
this process(developed by T. Macke; seeLarsen et al., 1993).
The secondary structure, and the few known tertiary inter-
actions, for thesesequenceswas deduced on the basisof pri-
mary and secondary structure homology with other Eucarya
235and 235-like rRNAs (Gutell et al., 1993).Secondarystruc-
ture diagrams were generated with the computer program
XRNA (developed by B. Weiserand H. Noller, University of
California, Santa Cruz).
Genomic DNA from the cultured lines of P. falciparum des-
ignated CAMP and 7G8 (Burkot et al., 1984) was isolated as
described previously (Li et al., 1993). Clones that contained
rDNA inserts from a plasmid library of mung bean nuclease
fragments of P. falciparum genomic DNA were initially se-
lected by hybridization to [32p]-labeled rRNA. The RNA was
isolated from a blood stage culture of P. falciparum and par-
tially cleaved with sodium borate prior to labeling at the 5'
position with ['Y-32P]ATP using polynucleotide kinase as de-
scribed (Dame & McCutchan, 1983). The complete gene set
was assembled by PCR with 7G8 genomic DNA as template
with the following pairs of primers:
903 (5'-CTTAACGATGGATGTCTTGG-3') and
580 (5'-CTTATTGCTTATCGGTATTGTTTGC-3').
873 (5'-ATAAGCCTCAACAGATCGTAAAAC-3') and
872 (5'-GCTTTAATTCTTTGTGAAAAAGGC-3').
Generally, the PCR reaction (100 ILL) contained 50 ng ge-
nomic DNA, 200 mM dNTP, and 2.5 mM MgC12 and 2.5 U
Taq DNA Polymerase with the buffer supplied by the man-
ufacturer (Perkin Elmer). The reaction was conducted in a
DNA Thermal Cycler (Perkin Elmer) with the following cy-
cles: 94 °C/l min, 50 °C/l min, 72 °C/2 min, for a total of 30
cycles. Amplified products were purified (Magic PCR preps.,
Promega) and cloned in pBluescript KS( -) (Stratagene) in the
SmaI site as described (Rogers et al., 1995). Cloning and other
general techniques were as described (Ausubel et al., 1992).
Inserts were sequenced in both directions by the dideoxy

11. M.J. Rogers et a[144
ACKNOWLEDGMENTS
We thank Dr. R.A. Wirtz and D. Seeley for some of the ma-
terials used in this study, and Dr. K.C. Rogers for critical
reading of the manuscript. This work was supported by the
NIH Intramural Research Program, and by NIH grant GM
48207 (awarded to R.G.). R.G. and S.D. also thank the W.M.
Keck Foundation for their generous support of RNA science
on the Boulder Campus.
ReceivedNovember 9, 1995; returned for revision December 14,
1995; revised manuscript receivedJanuary 22, 1996
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