RNA (1996), 2:134-145. Cambridge University Press. Printed in the USACopyright @ 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. McCUTCHAN11 Growth and Development Section, Laboratory of Parasitic Diseases. National Institute of Allergy and Infectious Diseases,

National Institutes of Health, Bethesda, Maryland 20892-0425, USA2 Department of MCD Biology. Campus Box 347. University of Colorado. Boulder, Colorado 80309-0347. USA3 Department of Chemistry and Biochemistry, Campus Box 215, University of Colorado, Boulder, Colorado 80309-0215, USA4 Laboratory of Parasitology, University of Leiden, Leiden, The Netherlands

ABSTRACT

The developmentally regulated transcription of at least two distinct sets of nuclear-encoded ribosomal RNAsis detected in Plasmodium species. The identification of functional differences between the two sets of rRNAsis 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. Analysisof the predicted secondary structures of the two units reveals the majority of differences between the A- and5-type genes occur in regions previously known to be variable. However, the predicted secondary structureof 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 geneis 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 withribosome function, in particular the GTPase site where the 5-type differs in a base pair and loop from all knownsequences. Further, the identification of compensatory changes at conserved points of interactions betweenthe 5.85-285 rRNAs indicates that transcripts from A- and 5-units should not be interchangeable.

Keywords: malaria; mosquito stage; ribosome; RNA secondary structure

Plasmodium falciparum is unusual in having rRNA genesets that are present in low copy number and dispersedon different chromosomes (McCutchan, 1986; Wellemset al., 1987; Li et al., 1994a). Unlike most eucaryotes,two distinct types of cytoplasmic Plasmodium 185 rRNAgenes have been shown to be developmentally reg-ulated, one type is predominantly expressed in themosquito host and the other in the mammalian host(Gunderson et al., 1987; Li et al., 1994b). Although theratio of the two types of transcripts changes dramati-cally during the developmental cycle of the parasite, itis likely that neither transcript disappears entirely atany point in the life cycle. Certainly there are extendedperiods of time when transcripts from both gene setsare present simultaneously (Gunderson et al., 1987;Waters et al., 1989). The sequence of two 185 rRNA

INTRODUCTION

Association between the rRNAs is likely to play an activerole in the assembly and interaction of the ribosomalsubunits (reviewed in Noller, 1991; Mitchell et al., 1992;Holmberg et al., 1994), and influences catalysis and ac-curacy in protein synthesis (reviewed in Noller et al.,1990, 1992). A typical eucaryote maintains homogeneouscopies of the 185-5.85-285 rRNA gene sets encoded inthe nucleus in the form of tandemly repeated clusters(Long & Dawid, 1980). The human malaria parasite

--

Reprint requests to: T.F. McCutchan, Growth and DevelopmentSection, 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

Structurally different rRNAs in Plasmodium 135

genes has been reported for P. falciparum (McCutchanet al., 1988). Here we describe the rRNA variation be-tween these two stage-specific types of ribosomes as itrelates to the 5.85 and 285 rRNAs.

The sequence of a 5.85 rRNA-intemal transcribedspacer 2 (IT52)-285 rRNA gene set from P. falciparumhas been reported, and expression of this gene set inthe blood stage has been 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 the5.85-285 rRNA gene set that is expressed in the sporo-zoite stage in the mosquito host (5-type). We describehere the isolation of a distinct 5.85-IT52-285 rRNAgene set from P. falciparum and demonstrate its expres-sion in the sporozoite stage. A detailed secondarystructure analysis shows that the 5-type 5.85 and 285rRNA gene set contains regions of both high conser-vation and regions with a high degree of variation withthe A-type gene set, and suggests possible functionaldifferences, inferring that genetic exchange betweenthe two types of rRNA units is restricted.

The 5.85 rRNA gene described here (Fig. 1) is almostidentical to the clone 119 described previously as oneof four P. falciparum 5.85 rRNA genes (5hippen-Lentzet al., 1990), with only a single A-+ C change at posi-tion 64. The 119 clone was not found to be expressedin the asexual stage (5hippen-Lentz et al., 1990). This5.85 rRNA gene shows only 80% homology with theasexually expressed 5.85 rRNA genes described previ-ously (5hippen-Lentz et al., 1990; Waters et al., 1995).The corresponding P. falciparum 5.85 rRNAs can thenbe clearly distinguished both by sequence and size (168nt for the gene in this study compared with 157-159 ntfor the asexually expressed genes). The IT52 (Fig. 1) is259 bp, compared with 197 bp for the asexually ex-pressed gene set. Apart from sharing an extreme AITbias (82%), the two IT52 sequences share little se-quence homology .The 5 nt at the 3' end of the IT52 areconserved between both P. falciparum gene sets andthree yeast species (van der 5ande et al., 19~2).

The 5' and 3' ends of the 285 rRNA encoded by thisgene set is inferred from secondary structure, phylo-genetic comparison, and sequence conservation withthe asexually expressed gene set (Waters et al., 1995).This corresponds to a 4. 17-kb 285 rRNA gene product,compared with 3.785-kb 285 rRNA for the asexuallyexpressed gene. The A and 5 genes are about 80% ho-mologous, although, as discussed below, the differ-ences are located primarily in variable regions that areeucaryote specific. Therefore, the 285 rRNAs of P. fal-ciparum are 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 sequence analysisthat had only 80% homology with the asexually ex-pressed 285 rRNA gene (Waters et al., 1995). It was iso-lated by hybridization of labeled rRNA to a genomiclibrary of mung bean nuclease-cleaved P. falciparumDNA (McCutchan et al., 1984; see the Materials andmethods). The sequence of this clone revealed thatabout 830 nt of the 5' end and about 500 nt of the 3' endof the corresponding 285 rRNA gene were missing(Fig. 1). The complete 5.85-IT52-285 gene set was thenassembled from overlapping fragments obtained fromPCR with genomic DNA as template. The primers forPCR were designed with oligonucleotides homologousto the 5' end of the P. falciparum 5.85 rRNA genes(5hippen-Lentz et al., 1990), and to a specific site in the5' end of the newly identified 285 rRNA gene (Fig. 1;see the Materials and methods). The 3' end of the 285rRNA gene was amplified with a primer homologousto a region specific to the newly identified 285 rRNAgene and one complementary to the conserved 3' endof the 285 rRNA genes. The identity of sequence over-lap between the PCR fragments and the genomic DNAfragment ensured assembly of the complete DNA se-quence. The sequence obtained overlapped with the185 rRNA-IT51-5.85 rRNA 5-type units from p; falcipa-rum (Rogers et al., 1995), and probably corresponds tothe pPfribl clone of P. falciparum rDNA identified pre-viously but not characterized in detail (Langsley et al.,1983).

Expression of the gene set in the sporozoite stage

We determined the pattern of expression of this distinct5.85-IT52-285 rRNA gene set during the life cycle of P.falciparum. By analogy with the expression of the 185rRNA genes of P. falciparum (McCutchan et al., 1988)and other Plasmodium species (Li et al., 1994a), thisgene set was suspected to be expressed in the mosquitohost upon differentiation of the parasite into thesporozoite stage. Therefore, RNA was isolated fromfour sources; purified sporozoites from Anophelesstephensi infected with P. falciparum, uninfected A.stephensi as a negative control, a blood-stage culture ofP. falciparum (Trager & Jensen, 1976), and RNA from anuninfected blood culture as a control.

The RNAs described above were used as templatefor RT/PCR with the complementary strand synthe-sized with primer 884 (specific to the gene identified inthis study) or 940, specific to the asexually expressedgene (Fig. 1). The PCR was then continued with 883 asthe second primer, conserved at the 5' end of both 285rRNA genes. The products of amplification were identi-fied by hybridization using oligonucleotides comple-mentary to specific regions of the P. falciparum 285rRNA genes (Fig. 2; see the Materials and methods).The asexually expressed gene hybridized to oligonu-

137Structurally different rRNAs in Plasmodium

RNA source RNA source

0-s0'"'

~+(\)0~0~"'

"1:!0O-

.c"1:!0)u~.s

s+0)

.~ oo .~~ &

"'o o

~ ~

~o0

-..c

+0--+

940 (A gene) 940 (A gene)884 (S gene) 884 (S gene)

cleotide 948 corresponding to a band of 580bp, whereasthe gene in this study hybridized to oligonucleotide 949as a band of 620 bp (see the Materials and methods).As expected, RT/PCR using blood-stage RNA as tem-plate confirmed the presence of transcripts from theasexually expressed 285 rRNA gene described previ-ously (Waters et al., 1995), with the same gene ex-pressed at a much lower level in RNA isolated frompurified sporozoites (Fig. 2). By RT/PCR from sporo-zoite RNA, a band of 620 bp was obtained that hybrid-ized to the probe (949; Fig. 2) specific for the new rRNA,but did not hybridize to a probe for the previously de-scribed A-type unit (probe 948). No PCR-derived bandcould be detected using RNA from uninfected mosquitosor with RNA from either infected blood stage cultureor uninfected blood (Fig. 2). No bands were obtainedby PCR from the RNA preparation without reversetranscriptase, demonstrating that the RNA is not con-taminated with genomic DNA (data not shown). Thisstudy determines that the new gene set from P. falcipa-rum is expressed in the sporozoite stage, and by con-vention it is therefore denoted as 5-type. The low levelof expression in the sporozoite stage of the asexuallyexpressed 285 rRNA gene is consistent with previous

data regarding the expression of the 185 rRNA genesof Plasmodium (Gunderson et al., 1987).

Association between the Plasmodium5.85 and 285 rRNAs sets

The 5.85 rRNA has a common evolutionary origin withthe 5' end of the 235 rRNA of eubacteria (Nazar, 1980;Jacq, 1981), and is known to base pair with the 5' endof the 285 rRNA (Pace et al., 1977; Noller et al., 1981).The presence of distinct p, falciparum 5.85 rRNA and285 rRNA genes of the A-type and 5-type suggestspossible unique interactions between the correspond-ing gene products of each type that, by analogy withother eucaryotic systems (Holmberg et al., 1994), mayhinder association between rRNA subunits of the othertype. A secondary structure prediction of the A-type5.85 rRNA with the 5' end of the A-type 285 rRNA andcomparison with that predicted for the 5-type rRNAsshows this to be the case (Fig. 3). In the base pairedstems that form between the 5.85 rRNA and the 285rRNA, there are substitutions that convert C-G basepairs in the A-type to U-A base pairs in the 5-type, andother changes to or from wobble base pairs (shaded re-

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 toduplicate filters with r2r]-labeled oligonucleotides 949 (5-specific) and 948 (A-specific). The first strand synthesis with RTwas carried out with either oligonucleotide 940 or 884, as described in the text. Arrow indicates the position of the 600-bpDNA marker, with the RNA source indicated above each lane.

Structurally different rRNAs in Plasmodium 139

TABLE 1. Differences between the P. falciparum A and S 5.85 and 285rRNA genes.

5.85 and 5'-half

of 285

3'-halfof 285 Total

236115526041193

7636

1214951156

12769383125

60

492058272245

10946142916133

2716632229111

-

Total number of differencesDifferences in base paired stems:

Compensatory changesNon-compensatory changes

1. Wobble pair2. Mismatched pair

Insertions or deletionsTransitionsTransversionsDifferences in loop regions:TransitionsTransversionsInsertionsDeletions

cussed below, the 5 gene is different at the GTPase site(positions 1059:1079 and 1084; Table 2) and suggests afunctional difference in the 5-type 285 rRNA. Theother substitutions occur at positions where functionalinformation is not known, so the significance of thesedifferences to the function of the 5-type ribosome is notunderstood. Interestingly, at a few positions (1346;1600; 2271), the 5 gene is more similar to the Eubacte-ria and/or Archaea consensus (Table 2). The 5-type 185rDNA units are usually more variable when compar-ing related Plasmodium species (Rogers et al., 1995; J.Li, unpubl. results), perhaps reflecting a higher muta-tion frequency in 5-type rDNA units. Information on5-type 285 rRNAs from other Plasmodium species willdetermine whether changes at these positions are ageneral feature of the genus.

The secondary structure of the 5-type and A-type285 rRNAs also provides information for the likely cat-alytic activity of the two types of ribosomes. TheGTPase activity inherent in the ribosome is located ina defined region in the 5' portion of the 285 rRNA, andthe corresponding part in eubacterial235 rRNA is alsothe site of interaction of a number of ribosomal proteinsand thiocillin antibiotics (Cundliffe, 1990; Douthwaiteet al., 1993). Interestingly, there is a compensatory basepair change and a change in the loop joining two heli-ces of this region in the 285 rRNA of the A-type and5-type (Fig. 5). The binding of ribosomal protein L11,the antibiotic thiostrepton, and the dependence ofmagnesium and ammonium ion on tertiary interactionshave been determined from mutants in this domain

Comparison of the A- and 5-type genes with regionsof the 5.85/285 rRNAs that are usually highly con-served in Eucarya is informative because it shows thatthe 5-type 285 rRNA is unusual at a number of posi-tions {Table 2). Of the 11 differences between A and 5genes at positions that are highly conserved in Eu-carya, the 5 gene is unusual at 8 positions {Table 2). Incontrast, the A gene 285 rRNA is uncommon at 4 ofthese 16 positions {positions 338; 1426; 2271; 2476 in E.coli numbering), whereas the A and 5 genes are bothunique 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

338856

1059107910841346

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

1346'1600 base pair

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 =00 78%

Status in Eubacterial, Archaea,and chloroplast phylogenetic

domains;" exceptionalfrequencies noted in bold

G in >95%A few A'sG ~ 99%, no A'sbC ~ 97%, no U'sbA ~ 99%, no U'sEub 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'sEub U ~ 99%, no A's;Arch G ~ 77%, no A'sEub 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%

a Y, pyrimidine; R, purine; Eub, eubacteria; Arch, archaea.b A few chloroplast have a 1059:1079 A:U base pair.c Except Aedes albopictus (mosquito) 1346:1600 A:U base pair

ne

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 IUGGUAGCA AC U CA CA

...G-C A

G G-C

A-UU-AG-C

FIGURE 5. The GTPase site of P. falciparum 285 rRNA. Secondarystructures 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 isshown, and differences with the A-type are indicated by arrows. Thesequence shown corresponds to nt 1618-1674 in the 5-type 285 rRNAand 1354-1410 in the A-type 285 rRNA.

AGUC

A

G

GUA

from E. coli 235 rRNA (Ryan et al., 1991; Lu & Draper,1994). The differences between the A- and 5-typeGTPase domains (Fig. 5) correspond to base pair 1059-1079 and 1084 in E. coli. The A-type rRNA resembles=98% of eucaryotes, Eubacteria, and Archaea with aG-C base pair, whereas the 5-type gene has an A-U inthis position, which, to date, is only seen in two chlo-roplast 235-like rRNAs (Gutell et al., 1993; 5.H. Dam-berger & R.R. Gutell, unpubl. alignments). Also, theA-type rRNA has an A at the position correspondingto 1084 that is seen in almost all eucaryotes and innearly all Eubacteria, Archaea, and chloroplast 235-likerRNAs (Table 2). The 5-type 285 rRNA is unique witha U at this position. Interestingly, the GI059-CI079:C1059-G1079 mutation in the E. coli GTPase domainreduces the binding of Lll and thiostrepton (Ryan et al.,1991). Mutation of AI084: U, which will mimic the 5-typerRNA (Fig. 5), also reduces Lll and thiostrepton binding(Ryan et al., 1991) and reduces the NH4+-dependenttm in the E. coli GTPase domain (Lu & Draper, 1994).There may then, as discussed below, be functional dif-ferences related to GTPase activity between the A-typeand 5-type 285 rRNAs. In contrast, the domain impli-cated in peptidyl transferase activity (Fig. 1) is conservedbetween the A-type and 5-type 285 rRNAs (Fig. 4B)and therefore any differences in tRNA selection andtranslational rates between the two molecules are likelyto be a consequence of differences outside this domain.

changes the character of the preexisting ribosome. Plas-modium species maintain 185-5.85-285 rRNAs with dif-ferent primary sequences differentially expressedduring development. Maintenance of variation withinthe central catalytic component of the ribosome (Nolleret al., 1992) of an organism would then seem to relateto function, although it is difficult to imagine the ben-efits to an organism of altering any of the catalytic ac-tivities known to be associated with rRNA. At least twopossibilities have precedence: (1) The rate of GTP hy-drolysis, including that associated with the ribosome,can playa central role in the control of growth and de-velopment. This ranges from the response to aminoacid starvation in bacteria, ribosome idling, and theproduction of "magic spot" in bacteria by the stringentresponse (Cashel & Rudd, 1987), to the association ofGTP hydrolysis associated with the ras gene productand the transformation and signaling in mammaliancells (Neer, 1995). (2) mRNA:ribosome association mayfavor translation of particular subsets of mRNAs (Chenet al., 1993) and potentially foster developmentalswitches.

Analysis of the secondary structures derived fromthe 5.85/285 rRNAs from the A-type and 5-type genesshows that the majority of differences occur in regionsof the rRNA where the consequences of these changesare unknown. However, a few A- and 5-type differ-ences are in conserved regions of the rRNA that may belinked to important functions during protein synthesis.We suggest characteristic differences in GTPase activitybetween the two types of ribosomes. Other evidence,discussed below, may also support the idea that ge-netic exchange between the types of units is restricted.

Variations on the conserved core of the GTPase sitehave been well studied for the E. coli GTPase domain(Ryan et al., 1991). Although not all of the correspond-ing differences between the A- and 5-type 285 rRNAsin the GTPase domains have been characterized in thebacterial system, perhaps the significance of thesechanges is that they identify a possible functional dif-ference in the two-ribosome system of P. falciparumthat can be quantified by the methods of Draper et al.(1993). In Plasmodium, the role of GTP hydrolysis inprotein synthesis may reflect a difference in syntheticneeds in the blood stage to the developing oocyst stageversus the relatively quiescent sporozoite. It also appearsthat interaction with the GTPase site of the ribosome isa biologically tolerated target for modulation of proteinsynthesis in response to slower rate of growth. Thus,these differences in the A and 5 genes in this regionof the rRNA may be functionally significant, althoughthe full significance of the sequence heterogeneities inthe A- and 5-type genes awaits experimental evidence.

Points of contact and interaction of rRNAs within theribosome are central to the assembly and function ofthe ribosome. We have determined the expression oftwo distinct 5.85 rRNA-285 rRNA gene sets in P. fal-

DISCUSSION

Members of the Plasmodium genus have ribosomeswhose rRNA content is developmentally regulated. Al-though some other organisms maintain heterogeneouspopulations of ribosomes (Etter et al., 1994), variationin these cases is probably based on the replacement ormodification of a preexisting ribosomal component that

""-Structurally different rRNAs in Plasmodium 143

chain termination method with oligonucleotides as sequenc-ing primers spaced approximately at 300-nt intervals. Se-quence alignments and other manipulations were with theLasergene software (DNASTAR) or Genetics ComputerGroup (GCG) sequence analysis software package. Nucleo-tide sequences reported in this paper have been reported toGenBank under the accession number 048228.

ciparum, and, by comparing the secondary structure ofeach, we find predictable differences in regions of therRNA associated with ribosome assembly through di-rect interaction. Compensatory changes associatedwith changes involved in Watson-Crick interactionsbetween the 5.85 rRNA and the 285 rRNA leads oneto believe that a positive advantage exists in maintain-ing structure and differences in these structures pre-vents total homogenization by genetic exchange .Interactions between the rRNAs of different units ap-pears to favor self-association (Fig. 4A,B). This is mostlikely to have the effect of limiting genetic exchange be-tween units; because rRNA processing and assemblyinto the ribosome is most likely coordinated, biologi-cal mixing at the RNA level is unlikely. Interactions in-volved in 5.85-285 rRNA base paired stems are provenphylogenetically and experimentally to be points of as-sociation (Pace et al., 1977; Gutell et al., 1994). Thecompensatory changes may reflect the necessity tomaintain the structure of the RNA with regard to itsrole 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) fromabout 0.5 mL of a blood stage culture of P. falciparum strain307 (Trager & Jensen, 1976) at approximately 5% parasitemia,and from about 464,000 purified sporozoites isolated from thesalivary glands of A. stephensi infected with P. falciparum strain307 (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 wasresuspended in a total of 50 p,L of RNase-free water and 10 p,Ltaken for analysis by RT/PCR with the Superscript Preampli-fication kit (GIBCO/BRL), with ONase I digestion and reversetranscription as described by the manufacturer. The reactionfrom the ONase I digestion was divided in two for reversetranscription with either oligonucleotide 884 (5'-CCCCCCTTAGTCCTGTG-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 byelectrophoresis 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'-GGA GATAATTCTATA TCGTA G-3') .

MATERIALS AND METHODS

Cloning and sequencing of the5.85-285 rRNA gl~ne set

Secondary structure analysis of the rRNAs

The secondary structure analysis of the P. falciparum 5.85 and285 A- and 5-type rRNAs was inferred from comparative se-quence analysis. These secondary structures are availablein postscript format from the World Wide Web site for RNAsecondary structures (Gutell, 1994); the URL for this site ishttp:/ /pundit.colorado.edu:8080/root.html. The secondarystructures are based on the paradigm that different RNA se-quences from the same RNA family (e.g., 285 rRNAs) willfold into a similar three-dimensional structure (Woese et al.,1983). Refinement of the secondary structure models for the235 and 235-like rRNAs is from the availability of nearly 300sequences (Gutell et al., 1993). The P. falciparum sequenceswere aligned with a large collection of previously aligned Eu-carya 235-like rRNA sequences, maintaining maximum pri-mary and secondary structure similarity (Woese et al., 1983;Gutell et al., 1985, 1994). The computer editor AE2 facilitatedthis process (developed by T. Macke; see Larsen et al., 1993).The secondary structure, and the few known tertiary inter-actions, for these sequences was deduced on the basis of pri-mary and secondary structure homology with other Eucarya235 and 235-like rRNAs (Gutell et al., 1993). Secondary struc-ture diagrams were generated with the computer programXRNA (developed by B. Weiser and H. Noller, University ofCalifornia, Santa Cruz).

Genomic DNA from the cultured lines of P. falciparum des-ignated CAMP and 7G8 (Burkot et al., 1984) was isolated asdescribed previously (Li et al., 1993). Clones that containedrDNA inserts from a plasmid library of mung bean nucleasefragments of P. falciparum genomic DNA were initially se-lected by hybridization to [32p]-labeled rRNA. The RNA wasisolated 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 setwas assembled by PCR with 7G8 genomic DNA as templatewith 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 UTaq DNA Polymerase with the buffer supplied by the man-ufacturer (Perkin Elmer). The reaction was conducted in aDNA 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 30cycles. Amplified products were purified (Magic PCR preps.,Promega) and cloned in pBluescript KS( -) (Stratagene) in theSma I site as described (Rogers et al., 1995). Cloning and othergeneral techniques were as described (Ausubel et al., 1992).Inserts were sequenced in both directions by the dideoxy

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 criticalreading of the manuscript. This work was supported by theNIH Intramural Research Program, and by NIH grant GM48207 (awarded to R.G.). R.G. and S.D. also thank the W.M.Keck Foundation for their generous support of RNA scienceon the Boulder Campus.

Received November 9, 1995; returned for revision December 14,1995; revised manuscript received January 22, 1996

REFERENCES

Ausubel FM, Brent R, Kingston RE, Moore DD, 5eidman JG, SmithJA, 5truhl K. 1992. Short protocols in molecular biology, 2nd ed. NewYork: Wiley-Interscience.

Burkot TR, Williams JL, 5chneider I. 1984. Infectivity to mosquitoesof Plasmodium falciparum clones grown in vitro from the same iso-late. Trans R Soc Trop Med Hyg 78:339-341.

Cashel M, Rudd KE. 1987. The stringent response. In: Neidhardt FC,Ingraham JL, Low KB, Magasanik B, 5chaechter M, UmbargerHE, eds. Escherichia coli and Salmonella typhimurium. Cellular andmolecular biology. Washington, DC: American Society for Micro-biology. pp 1410-1438.

Chen CY, Macejak DG, Oh 5K, 5arnow P. 1993. Translation initia-tion by internal ribosome binding of eukaryotic rnRNA molecules.In: Nierhaus KH, Franceschi F, 5ubramanian AR, Erdmann VA,Wittmann-Liebold B, eds. The translational apparatus: Structure,function, regulation, evolution. New York: Plenum Press. pp 229-240.

Cundliffe E. 1990. Recognition sites for antibiotics within rRNA. In:Hill WE, Dahlberg A, Garret RA, Moore PB, 5chlessinger D,Warner JR, eds. The ribosome: Structure, function, & evolution. Wash-ington, DC: American Society for Microbiology. pp 479-490.

Dame JB, McCutchan TF. 1983. The four ribosomal DNA units of themalaria parasite Plasmodium berghei. J Bioi Chem 258:6984-6990.

Douthwaite 5, Vester B, Aagaard C, Rosendahl G. 1993. Antibioticand protein interactions with the GTPase and peptidyl transfer-ase regions in 235 rRNA. In: Nierhaus KH, Franceschi F,5ubramanian AR, Erdmann VA, Wittmann-Liebold B, eds. Thetranslational apparatus. New York: Plenum Press. pp 339-346.

Draper DE, Deckeman IC, Vartikar JV. 1993. Physical studies of ri-bosomal protein-RNA interactions. Methods EnzymoI164:203-220.

Enea V, Corredor V. 1991. The evolution of plasmodial stage-specificrRNA genes is dominated by gene conversion. J Mol Evol 32 :183-186.

Etter A, Bernard V, Kenzelmann M, Tobler H, Muller F. 1994. Ribo-somal heterogeneity from chromatin diminution in Ascaris lum-bricoides. Science 265:954-956.

Gunderson JH, 5ogin ML, Wollet G, Hollingdale M, De La Cruz VF,Waters AP, McCutchan TF. 1987. Structurally distinct, stage-specific ribosomes occur in Plasmodium. Science 238:933-937.

Gutell RR. 1994. Collection of small subunit (165- and 235-like) ri-bosomal RNA structures: 1994. Nucleic Acids Res 22:3502-3507.

Gutell RR, Gray MW, 5chnare MN. 1993. A compilation of large sub-unit (235 and 235-like) ribosomal RNA structures. Nucleic AcidsRes 21:3055-3074.

Gutell RR, Larsen N, Woese CR. 1994. Lessons from an evolvingrRNA: 165 and 235 rRNA structures from a comparative perspec-tive. Microbiol Rev 58:10-26.

Gutell RR, Weiser B, Woese CR, Noller HF. 1985. Comparative anat-omy of 165-like ribosomal RNA. Prog Nucleic Acid Res Mol Bioi32:155-216.

Holmberg L, Melander Y, NygArd 0. 1994. Probing the conforma-tional changes in 5.85, 185 and 285 rRNA upon association of de-rived subunits into complete 805 ribosomes. Nucleic Acids Res22:2776-2783.

Jacq B. 1981. Sequence homologies between eukaryotic 5.85 rRNAand the 5' end of prokaryotic 235 rRNA: Evidence for a commonevolutionary origin. Nucleic Acids Res 9:2193-2933.

Langsley G, Hyde IE, Goman M, Scaife IG. 1983. Cloning and char-acterisation of the rRNA genes from the human malaria parasitePlasmodium falciparum. Nucleic Acids Res 11 :8703-8717.

Larsen N, Olsen GI, Maidak BL, McCaughey MI, Overbeek R, MackeTI, Marsh TL, Woese CR. 1993. The ribosomal database project.Nucleic Acids Res 21:3021-3023.

Li I, McConkey GA, Rogers MI, Waters AP, McCutchan TF. 1994a.Plasmodium: The developmentally regulated ribosome. Exp Par-asitol 78:437-441.

Li I, Wirtz RA, McConkey GA, Sattabongkot I, McCutchan TF. 1994b.Transition of Plasmodium vivax ribosome types corresponds tosporozoite differentiation in the mosquito. Mol Biochem Parasitol65:283-289.

Li I, Wirtz RA, Schneider I, Muratova OV, McCutchan TF, AppiahA, Hollingdale MR. 1993. Plasmodium falciparum: Stage-specific ri-bosomal RNA as a potential target for monitoring parasite devel-opment in Anopheles stephensi. Exp Parasitol 76:32-38.

Long EO, Dawid IB. 1980. Repeated genes in eukaryotes. Annu RevBiochem 49:727-764.

Lu M, Draper DE. 1994. Bases defining an ammonium and magne-sium ion-dependent tertiary structure within the large subunit ri-bosomal RNA. J Mol BioI 244:572-585.

McCutchan TF. 1986. The ribosomal genes of Plasmodium. lnt RevCytol 99:295-309.

McCutchan TF, de la Cruz VF, Lal AA, Gunderson IH, Elwood HI,Sogin ML. 1988. Primary sequences of two small subunit ribo-somal RNA genes from Plasmodium falciparum. Mol Biochem Par-asitol 28:63-68.

McCutchan TF, Hansen IL, Dame IB, Mullins IA. 1984. Mung beannuclease cleaves Plasmodium DNA at sites before and after genes.Science 225:625-628.

McCutchan TF, Li I, McConkey GA, Rogers MI, Waters AP. 1995. Thecytoplasmic ribosomal RNAs of Plasmodium spp. Parasitol Today11:134-138.

Mitchell P, Osswald M, Brimacombe R. 1992. Identification of inter-molecular cross-links at the subunit interface of the Escherichia coliribosome. Biochemistry 31 :3004-3011.

Nazar RN. 1980. A 5.8 S rRNA-Iike sequence in prokaryotic 23 SrRNA. FEBS Lett 119:212-214.

Neer El. 1995. Heterotrimeric G proteins: Organizers of transmem-brane signals. Cell 80:249-257.

Noller HF. 1991. Ribosomal RNA and translation. Annu Rev Biochem60:191-227.

Noller HF, Hoffarth V, Zimniak L. 1992. Unusual resistance of pep-tidyl transferase to protein extraction methods. Science 256:1416-1419.

Noller HF, Kop IA, Wheaton V, Brosius I, Gutell RR, Kopylov AM,Dohme F, Herr W, Stahl DA, Gupta R, Woese CR. 1981. Second-ary structure model for 235 ribosomal RNA. Nucleic Acids Res22:6167-6189.

Noller HF, Moazad D, Stern S, Powers T, Allen PN, Robertson IM,Weiser B, Triman K. 1990. Structure of rRNA and its functionalinteractions in translation. In: Hill WE, Dahlberg A, Garret RA,Moore PB, Schlessinger D, Warner IR, eds. The ribosome: Struc-ture, function, & evolution. Washington, DC: American Society forMicrobiology. pp 73-92.

Pace NR, Walker TR, Schroeder E. 1977. Structure of the 5.85 RNAcomponent of the 5.85-285 ribosomal RNA junction complex. Bio-chemistry 16:5321-5328.

Rogers MI, McConkey GA, Li I, McCutchan TF. 1995. The ribosomalDNA loci in Plasmodium fakiparum accumulate mutations indepen-dently. J Mol BioI 254:881-891.

Ryan PC, Lu M, Draper DE. 1991. Recognition of the highly con-served GTPase center of 235 ribosomal RNA by ribosomal pro-tein L11 and the antibiotic thiostrepton. J Mol BioI 221:1257-1268.

SantaLucia I Ir, Kierzek R, Turner DH. 1991. Stabilities of consecu-tive A.C, C.C, G.G, U.C, and U.U mismatches in RNA inter-nalloops: Evidence for stable hydrogen-bonded U .U and C .C +pairs. Biochemistry 30:8242-8251.

Shippen-Lentz D, Afroze T, Vezza AC. 1990. Heterogeneity and ex-pression of the Plasmodium falciparum 5.85 ribosomal RNA genes.Mol Biochem ParasitoI38:113-120.

Trager W, Iensen IB. 1976. Human malaria parasites in continuousculture. Science 193:673-675.

Unnasch TR, McLafferty M, Wirth DF. 1985. The primary structure

Structurally different rRNAs in Plasmodium 145

subunit rRNA expressed in blood stages of Plasmodium falciparum.Mol Biochem Parasitol 72:227-237.

Wellems TE, Walliker D, Smith CL, Do Rosario YE, Maloy WL, How-ard RJ, Carter R, McCutchan IF. 1987. A histidine-rich proteingene marks a linkage group favored strongly in a genetic crossof Plasmodium falciparum. Cell 49:633-642.

Woese CR, Gutell R, Gupta R, Noller HF. 1983. Detailed analysis ofthe higher-order structure of 16s-like ribosomal ribonucleic acids.Microbiol Rev 47:621-669.

of the rRNA insertions of Plasmodium lophurae. Mol Biochem Par-asitoI16:149-161.

van der Sande CAFM, Kwa M, van Nues RW, van Heerikhuizen H,Raue HA, Planta RJ. 1992. Functional analysis of internal tran-scribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA. J MolBioI 223:899-910.

Waters AP, Syin C, McCutchan IF. 1989. Development regulation ofstage-specific ribosome populations in Plasmodium. Nature 342:438-440.

Waters AP, White W, McCutchan IF. 1995. The structure of the large