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Gutell 055.rna.1996.02.0134


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Gutell 055.rna.1996.02.0134

  1. 1. 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,1ANDREW P. WATERS,4 and THOMAS F. McCUTCHAN11Growth 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 NetherlandsABSTRACTThe 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 structurePlasmodiumfalciparumis unusual in having rRNA genesetsthat arepresentin low copy number and dispersedon different chromosomes(McCutchan, 1986;Wellemset al., 1987;Li et al., 1994a).Unlike most eucaryotes,two distinct types of cytoplasmicPlasmodium185rRNAgenes 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 cycleof the parasite, itis likely that neither transcript disappears entirely atany point in the life cycle. Certainly there areextendedperiods of time when transcripts from both gene setsare present simultaneously (Gunderson et al., 1987;Waters et al., 1989).The sequence of two 185 rRNAINTRODUCTIONAssociationbetweentherRNAs islikely to play anactiverole in the assembly and interaction of the ribosomalsubunits (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 typicaleucaryotemaintainshomogeneouscopiesof the 185-5.85-285rRNA genesetsencoded 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;
  2. 2. Structurally different rRNAs in Plasmodium 135geneshasbeen reported for P.falciparum(McCutchanet al., 1988).Here we describethe rRNA variation be-tween thesetwo stage-specifictypes of ribosomes asitrelates to the 5.85 and 285 rRNAs.The sequenceof a 5.85 rRNA-intemal transcribedspacer2 (IT52)-285 rRNA gene set from P.falciparumhas been reported, and expression of this gene set inthe 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 the5.85-285rRNA genesetthat is expressedin the sporo-zoite stagein the mosquito host (5-type). We describehere the isolation of a distinct 5.85-IT52-285 rRNAgenesetfrom P.falciparumand 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 regionswith ahigh degreeof variation withthe A-type gene set, and suggestspossible functionaldifferences, inferring that genetic exchange betweenthe two types of rRNA units is restricted.The 5.85 rRNA genedescribedhere (Fig. 1)is almostidentical to the clone 119described previously as oneof four P.falciparum5.85 rRNA genes(5hippen-Lentzet al., 1990),with only a single A-+ C change at posi-tion 64. The 119clone was not found to be expressedin the asexualstage(5hippen-Lentz et al., 1990).This5.85 rRNA gene shows only 80% homology with theasexually expressed5.85 rRNA genesdescribedprevi-ously (5hippen-Lentz et al., 1990;Waters et al., 1995).The corresponding P.falciparum5.85 rRNAs can thenbe clearly distinguished both by sequenceand size(168nt for the genein this study compared with 157-159ntfor the asexually expressedgenes).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-quencehomology .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 sequenceconservation withthe asexually expressedgene set (Waters et al., 1995).This correspondsto a4.17-kb285 rRNA gene product,compared with 3.785-kb 285 rRNA for the asexuallyexpressedgene. The A and 5 genesare about 80%ho-mologous, although, as discussed below, the differ-encesarelocated primarily in variable regions that areeucaryote specific. Therefore, the 285 rRNAs of P.fal-ciparumare distinct in both length and sequence.RESULTSThe sequem~e of a distinct 5.8S-ITS2-28S gem~ setAn rDNA clone was identified by sequenceanalysisthat had only 80% homology with the asexually ex-pressed285rRNA gene(Waterset 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;seethe Materials andmethods). The sequence of this clone revealed thatabout 830nt of the 5 end and about 500nt of the 3 endof the corresponding 285 rRNA gene were missing(Fig. 1). Thecomplete 5.85-IT52-285genesetwas thenassembledfrom overlapping fragments obtained fromPCRwith genomic DNA astemplate. The primers forPCRwere designedwith oligonucleotideshomologousto the 5 end of the P. falciparum 5.85 rRNA genes(5hippen-Lentz et al., 1990),and to a specificsite in the5 end of the newly identified 285 rRNA gene (Fig. 1;seethe 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 285rRNA genes.The identity of sequenceover-lap between the PCRfragments and the genomic DNAfragment ensured assemblyof the complete DNA se-quence. The sequenceobtained overlapped with the185rRNA-IT51-5.85 rRNA 5-type units from p; falcipa-rum (Rogerset al., 1995),and probably corresponds tothe pPfribl clone of P.falciparumrDNA identified pre-viously but not characterizedin detail (Langsley et al.,1983).Expression of the gene set in the sporozoite stageWedetermined the pattern of expressionof this distinct5.85-IT52-285rRNA gene setduring the life cycleof P.falciparum.By analogy with the expression of the 185rRNA genes of P.falciparum(McCutchan et al., 1988)and other Plasmodiumspecies (Li et al., 1994a),thisgenesetwas suspectedto be expressedin the mosquitohost upon differentiation of the parasite into thesporozoite stage. Therefore, RNA was isolated fromfour sources; purified sporozoites from Anophelesstephensiinfected with P. falciparum, uninfected A.stephensiasa negative control, ablood-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(specificto the geneidentified inthis study) or 940, specific to the asexually expressedgene(Fig. 1). The PCRwas then continued with 883asthe secondprimer, conserved at the 5 end of both 285rRNA genes.Theproducts of amplification were identi-fied by hybridization using oligonucleotides comple-mentary to specific regions of the P. falciparum 285rRNA genes (Fig. 2; seethe Materials and methods).The asexually expressed gene hybridized to oligonu-
  3. 3. 137Structurally different rRNAs in PlasmodiumRNA source RNA source0-s0"~+()0~0~""1:!0O-.c"1:!0) oo .~~ &"o o~ ~~o0-..c+0--+940 (A gene) 940 (A gene)884 (S gene) 884(S gene)cleotide948corresponding to aband of 580bp, whereasthe genein this study hybridized to oligonucleotide 949as a band of 620bp (seethe Materials and methods).As expected, RT/PCRusing blood-stage RNA astem-plate confirmed the presence of transcripts from theasexually expressed285 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/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-derivedbandcouldbedetectedusingRNA from uninfectedmosquitosor 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 genesetfrom P.falcipa-rum is expressedin the sporozoite stage, and by con-vention it is therefore denoted as5-type. The low levelof expression in the sporozoite stage of the asexuallyexpressed285 rRNA gene is consistent with previousdata regarding the expression of the 185 rRNA genesof Plasmodium(Gunderson et al., 1987).Association between the Plasmodium5.85 and 285 rRNAs setsThe 5.85 rRNA has acommon evolutionary origin withthe 5 end of the 235 rRNA of eubacteria(Nazar, 1980;Jacq,1981),and is known to basepair with the 5 endof the 285 rRNA (Paceet al., 1977;Noller et al., 1981).The presence of distinct p, falciparum5.85 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 associationbetween rRNA subunits of the othertype. A secondary structure prediction of the A-type5.85 rRNA with the 5 end of the A-type 285rRNA 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 basepairs in the 5-type, andother 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 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.
  4. 4. Structurally different rRNAs in Plasmodium 139TABLE 1. Differences between the P. falciparum A and S 5.85 and 285rRNA genes.5.85 and 5-halfof 2853-halfof 285 Total236115526041193763612149511561276938312560492058272245109461429161332716632229111-Total number of differencesDifferences in base paired stems:Compensatory changesNon-compensatory changes1. Wobble pair2. Mismatched pairInsertions or deletionsTransitionsTransversionsDifferences in loop regions:TransitionsTransversionsInsertionsDeletionscussedbelow, the 5 geneis different at the GTPasesite(positions 1059:1079and 1084;Table2) and suggestsafunctional difference in the 5-type 285 rRNA. Theother substitutions occur at positions where functionalinformation is not known, so the significance of thesedifferencesto the function of the 5-type ribosomeis notunderstood. 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 185rDNA 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 on5-type 285 rRNAs from other Plasmodiumspecieswilldetermine whether changes at these positions are ageneral feature of the genus.The secondary structure of the 5-type and A-type285 rRNAs alsoprovides information for the likely cat-alytic activity of the two types of ribosomes. TheGTPaseactivity 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 siteof interaction of anumber of ribosomal proteinsand thiocillin antibiotics (Cundliffe, 1990;Douthwaiteet al., 1993).Interestingly, there is acompensatorybasepair change and achange 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 domainComparison of the A- and 5-type geneswith regionsof the 5.85/285 rRNAs that are usually highly con-served in Eucaryais informative becauseit shows thatthe 5-type 285 rRNA is unusual at a number of posi-tions {Table2). Of the 11differences between A and 5genes at positions that are highly conserved in Eu-carya, the 5 gene is unusual at 8 positions {Table2). Incontrast, the A gene 285 rRNA is uncommon at 4 ofthese16positions {positions 338;1426;2271;2476in E.colinumbering), 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 conservedPosition(E. coli numbering)~Status in Eucarya Comments3388561059107910841346P. falciparumA gene SgeA GC AG AC UA UG AG ~ 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 pair1059:1079 base pair1059:1079 base pair13461600basepairG Rarely a U; G := 75%c u Only Eucarya with a Uc; C = 99% 1346:1600 base pair2271 u A Only Eucarya with a U; A =0078%Status in Eubacterial, Archaea,and chloroplast phylogeneticdomains;" exceptionalfrequencies noted in boldG in >95%A few AsG ~ 99%, no AsbC ~ 97%, no UsbA ~ 99%, no UsEub A ~ 42%, G = 55%;Arch G ~ 100%Eub G ~ 93%, a few Us;Arch G = 100%Eub U ~ 42%, C = 55%;Arch C = 100%Eub G ~ 96%, no Us;Arch A ~ 65%, no UsEub U ~ 99%, no As;Arch G ~ 77%, no AsEub A ~ 100%, no Ys;Arch A ~ 70%, C ~ 30%2419 G A Only Eucarya with an A; G ==99% 2397:2419 base pair2476 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 pairne
  5. 5. M.J. Rogers et al.142AtC UUAAUt C-G AGU GU,AA UCG G GU AI. I I I I I IUGGUAGCA AC U CA CA...G-C AG G-CA-UU-AG-CFIGURE 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.AGUCAGGUAfrom E. coli235 rRNA (Ryan et al., 1991;Lu & Draper,1994). The differences between the A- and 5-typeGTPasedomains (Fig. 5) correspond to basepair 1059-1079and 1084in E. coli. The A-type rRNA resembles=98% of eucaryotes, Eubacteria, and Archaea with aG-C basepair, whereas the 5-type genehasan A-U inthis 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, theA-type rRNA has an A at the position correspondingto 1084that is seen in almost all eucaryotes and innearly all Eubacteria,Archaea,and chloroplast 235-likerRNAs (Table2). The 5-type 285 rRNA is unique witha U at this position. Interestingly, the GI059-CI079:C1059-G1079mutation in the E. coli GTPasedomainreducesthebinding of Lll and thiostrepton (Ryanetal.,1991).Mutation ofAI084:U, which will mimic the 5-typerRNA (Fig.5),alsoreducesLll andthiostrepton binding(Ryan et al., 1991)and reduces the NH4+-dependenttmin the E. coli GTPasedomain (Lu & Draper, 1994).There may then, asdiscussedbelow, be functional dif-ferencesrelated to GTPaseactivity between the A-typeand 5-type 285 rRNAs. In contrast, the domain impli-catedin peptidyl transferaseactivity (Fig. 1)isconservedbetween the A-type and 5-type 285 rRNAs (Fig. 4B)and therefore any differences in tRNA selection andtranslationalratesbetweenthe two moleculesarelikelyto be aconsequenceof differencesoutside this domain.changesthe characterof the preexisting ribosome. Plas-modiumspeciesmaintain 185-5.85-285rRNAs with dif-ferent primary sequences differentially expressedduring development. Maintenance of variation withinthe central catalyticcomponent of the ribosome (Nolleret al., 1992)of an organism would then seemto 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 associatedwith rRNA. At leasttwopossibilities 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 aminoacid starvation in bacteria, ribosome idling, and theproduction of "magic spot" in bacteriaby the stringentresponse (Cashel & Rudd, 1987),to the association ofGTP hydrolysis associatedwith the rasgene productand the transformation and signaling in mammaliancells(Neer, 1995).(2)mRNA:ribosome associationmayfavor translation of particular subsetsof mRNAs (Chenet al., 1993) and potentially foster developmentalswitches.Analysis of the secondary structures derived fromthe 5.85/285rRNAs from the A-type and 5-type genesshows that the majority of differences occur in regionsof the rRNA where the consequencesof thesechangesare unknown. However, a few A- and 5-type differ-encesarein conservedregions of the rRNA that may belinked to important functions during protein synthesis.We suggestcharacteristicdifferencesin GTPaseactivitybetween 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 GTPasesitehave 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 rRNAsin the GTPasedomains have been characterizedin thebacterial system, perhaps the significance of thesechangesis 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 syntheticneedsin the blood stageto the developing oocyst stageversusthe relatively quiescentsporozoite.It alsoappearsthat interaction with the GTPasesite of the ribosome isabiologically 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 sequenceheterogeneities inthe A- and 5-type genesawaits experimental evidence.Points of contactand 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-DISCUSSIONMembers of the Plasmodiumgenus have ribosomeswhose rRNA content is developmentally regulated. Al-though someother organisms maintain heterogeneouspopulations of ribosomes (Etter et al., 1994),variationin thesecasesis probably basedon the replacement ormodification of apreexisting ribosomalcomponent that
  6. 6. ""-Structurally different rRNAs in Plasmodium 143chain termination method with oligonucleotides assequenc-ing primers spaced approximately at 300-nt intervals. Se-quence alignments and other manipulations were with theLasergene software (DNASTAR) or Genetics ComputerGroup (GCG) sequenceanalysis software package. Nucleo-tide sequencesreported in this paper have been reported toGenBank under the accessionnumber 048228.ciparum,and, by comparing the secondarystructure ofeach,we find predictable differences in regions of therRNA associatedwith ribosome assemblythrough 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 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 mostlikely to have the effectof limiting geneticexchangebe-tween units; becauserRNA processing and assemblyinto the ribosome is most likely coordinated, biologi-cal mixing at the RNA level is unlikely. Interactions in-volved in 5.85-285rRNA basepaired stemsareprovenphylogenetically 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 rRNATotal 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. stephensiinfected 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,Lof 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 METHODSCloning and sequencing of the5.85-285 rRNA gl~ne setSecondary structure analysis of the rRNAsThe secondarystructure analysisof the P.falciparum5.85 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:/ / The secondarystructures arebasedon the paradigm that different RNA se-quences from the same RNA family (e.g., 285 rRNAs) willfold into a similar three-dimensional structure (Woeseet 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.falciparumsequenceswere 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 facilitatedthis 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 Eucarya235and 235-like rRNAs (Gutell et al., 1993).Secondarystruc-ture diagrams were generated with the computer programXRNA (developed by B. Weiserand 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 5position 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) and580 (5-CTTATTGCTTATCGGTATTGTTTGC-3).873 (5-ATAAGCCTCAACAGATCGTAAAAC-3) and872 (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 theSmaI 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
  7. 7. M.J. Rogers et a[144ACKNOWLEDGMENTSWe 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.ReceivedNovember 9, 1995; returned for revision December 14,1995; revised manuscript receivedJanuary 22, 1996REFERENCESAusubel FM, Brent R, Kingston RE, Moore DD, 5eidman JG, SmithJA, 5truhl K. 1992.Shortprotocolsin molecularbiology,2nded.NewYork: Wiley-Interscience.Burkot TR, Williams JL, 5chneider I. 1984.Infectivity to mosquitoesof Plasmodiumfalciparumclonesgrown in vitro from the sameiso-late. TransR SocTropMed Hyg 78:339-341.CashelM, Rudd KE. 1987.The stringent response.In: Neidhardt FC,Ingraham JL, Low KB, Magasanik B, 5chaechter M, UmbargerHE, eds. 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