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    Gutell 022.mbp.1992.52.0075 Gutell 022.mbp.1992.52.0075 Document Transcript

    • Molecular and Biochemical Parasitology, 52 (1992) 75-84 75© 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00MOLBIO 01719Characterization of the rDNA unit and sequence analysis of the smallsubunit rRNA and 5.8S rRNA genes from Tritrichomonasfoetus*Debopam Chakrabarti 12, John B. Dame 1., Robin R. Gutell 3, and Charles A. Yowell 11Department of lnfectious Diseases, College of Veterinary Medicine, and 2Interdisciplinary CenterJbr Biotechnology Research,University ~?fFlorida, Gainesville, FL, USA; and 3MCD Biology, University of Colorado, Boulder, CO, USA(Received 6 September 1991 ; accepted 26 November 1991)The ribosomal RNA gene unit of the protozoan parasite Tritrichomonasfoetus has been cloned and analyzed. Southernblot analysis of the genomic DNA showed that the ribosomal RNA gene unit is organized as a tandem head to tail repeat witha unit length of 6 kb. By Northern analysis a primary transcript of 5.8 kb was detected. Copy number analysis showed thepresence of 12 copies of the ribosomal RNA gene unit. The lengths of the small subunit ribosomal RNA and 5.8S ribosomalRNA are 1571 bp and 159 bp, respectively, as determined by sequence analysis. The T.foetus small subunit ribosomal RNAsequence is one of the shortest eukaryotic small subunit rRNA sequences, similar in length to those from 2 otheramitochondrial protists. Although shorter than the majority of the eukaryotic small subunit ribosomal RNAs, this sequencemaintains the primary and secondary structure common to all eukaryotic small subunit ribosomal RNA structures, whiletruncating sequences found within the eukaryotic variable regions. The length of the large subunit ribosomal RNA wasmeasured at 2.5 kb.Key words: Trichomonad; Ribosomal RNA; 16S rRNA; 18S rRNA; Eukaryote; ProtozoaIntroductionThe flagellated, anaerobic protozoan para-site Tritrichomonasfoetus is the causative agentof bovine trichomoniasis, a contagious sexu-ally transmitted disease characterized byinfertility, abortion and pyometra in cattleCorrespondence address: John B. Dame, Department ofInfectious Diseases, College of Veterinary Medicine, Univer-sity of Florida, Gainesville, FL 32611, USA.Note. Nucleotide sequence data reported in this paper havebeen submitted to the GenBankTM data base with the accessionnumber M81842.This paper is part of the University of Florida AgriculturalExperiment Station Journal Series, No. R-01860.Abbreviations." rRNA, ribosomal RNA; rDNA unit, ribosomalRNA gene unit; SSrRNA, small subunit rRNA; ITS, internaltranscribed spacer; CHEF, contour-clamped homogeneouselectric field; CsTFA, cesium trifluoroacetic acid; TBE, Tris-borate-EDTA buffer.[1]. Basic to control of the disease is a sensitiveand specific diagnostic test. We have initiated astudy of the rRNA genes of this organism in aneffort to develop molecular markers for thediagnosis of infection. The rDNA units in mosteukaryotes studied to date encode 18S, 5.8Sand 28S rRNAs and are tandemly repeatedwith the number of copies varying fromapprox. 100-5000 [2]. Although these featuresof ribosomal RNA gene units are wellconserved in most eukaryotes, the mostnotable exceptions to the general rule of thesupramolecular organization are found inprotozoa. In some protozoan parasites thenumber of rDNA repeats are often remarkablylow and are not tandemly arranged such asBabesia with 3 copies [3] and Plasmodium with4-8 copies [4-6]. The length of rDNA unitsvary widely, from 5.5 kb in Giardia intestinalis[7] to 44 kb in rat [8]. The SSrRNA also differsin length to its currently known extremeswithin the protozoa from 1244 bp in Vair-
    • 76imorpha necatrix to 2319 bp in Trypanosomacruzi [9]. We report in this paper the cloningand characterization of the rDNA unit from T.,foetus. The SSrRNA in this organism is one ofthe shortest reported and the copy number ofthe rDNA repeating unit is low, but tandemlyarranged.Materials and MethodsT. foetus cultures were grown in TYMmedium, pH 7.0, supplemented with 5% heatinactivated calf serum [10]. Strains Crop-I(kindly provided by J.M. Cheney, ColoradoState University) and UT-I (American TypeCulture Collection #30233) were used through-out these studies. Organisms were harvested inthe mid-log growth phase.Genomic DNA and total RNA wereroutinely isolated by extracting cells with asolution containing 4 M guanidinium isothio-cyanate/5 mM sodium citrate/ 10 mM EDTA/0.5% N-lauryl sarcosine /1 mM 2-mercapto-ethanol, followed by fractionation via isopyc-nic gradient centrifugation in cesium trifluoro-acetate (CsTFA) [11]. Total nucleic acids wereisolated by phenol-chloroform extraction fromcells incubated at 65°C for 2 h in extractionbuffer containing 0.1 M NaC1/ 0.025 Msodium EDTA/ 0.05 M Tris-HC1, pH 8.0/1% sodium dodecyl sulfate/ 1 mg ml 1proteinase K [12]. Intact chromosomal DNAwas prepared in 1% agarose blocks asdescribed [13]. Plasmid DNA was isolated bythe boiling method [12]. Genomic and plasmidDNAs were restriction enzyme digested, elec-trophoresed on agarose gels, and blotted onnylon membranes as described [12]. TotalRNA was fractionated in formaldehyde agar-ose gels [12] adjacent to a 0.24-9.5-kb RNAladder (Gibco/BRL, Gaithersburg, MD), andblotted on nylon membranes.ResultsIn an effort to identify the rDNA cistron inT..foetus, genomic DNA samples from 2different isolates, Crop-1 and UT-1, weredigested to completion with various restric-tion enzymes. Southern blots of these digestswere probed with 5-end labeled total RNA. Ascan be seen from Fig. 1A, digestion of T.foetusgenomic DNA, derived from both Crop-1 andUT-1, with PvulI, Smal and XhoI gave rise to asingle 6-kb band indicating the presence of asingle site in a repeating unit whereas digestionwith SstI resulted in 2 bands of 5.2 kb and 0.8kb. The faint bands around 1.1 kb in the Smaldigest, and > 10 kb in the PvulI and Sstldigests, may represent the end fragment of therepeat. End fragments were not identified inthe XhoI digest indicating that the flankingfragments may be very large or they bothcoincidentally have sizes similar to the rDNAunit length. The organization of rDNArepeating unit is shown in Fig. 1C.T. foetus genomic DNA was digested tocompletion with XhoI, separated on a 0.8%agarose gel, and 6-kb DNA fragments wereelectrophoresed onto DEAE cellulose mem-brane. The isolated fragments were ligated intothe XhoI site of pBluescript SK(+) followedby transformation into XL-1 blue cells.Transformed colonies were screened with 32p_labeled total RNA probe prepared as described[14]. The DNA was isolated from positivecolonies, digested with XhoI and probed with5-end labeled RNA probes for further con-firmation. A positive clone containing the 6-kbfragment, designated as p433, was used infurther studies. A physical map of the T. foetusrDNA unit was constructed by digestion withdifferent restriction enzymes alone or invarious combinations and also from sequenceanalysis (Fig. 1D).Northern blot analysis of the T. Joetus totalRNA with labeled p433 (Fig. 1B) indicates thatthe length of the large and small subunitrRNAs are approximately 2.5 and 1.6 kb,respectively. This suggests that the T../oetusrRNAs are smaller than the majority ofeukaryotes studied to date [9]. Two largermolecular length bands of 5.8 kb and 4 kb werealso detected upon overexposure of the filmwhich are thought to be precursor rRNAs. The5.8-kb rRNA precursor is long enough to be
    • the primary transcript, suggesting that the non-transcribed spacer is approximately 200 bplong. The 4-kb transcript is most likelygenerated from the primary transcript byprocessing cleavages but the processing siteswhich give rise to it are not known.A combination of sequencing strategies wereused to obtain the nucleotide sequence of theSSrRNA coding region. Since many of theuniversal 18S rRNA sequencing primers [15]did not work, the information from therestriction map was used to prepare exonu-clease Ill deletion subclones. In additionspecific sequencing primers were preparedusing the sequence obtained from the sub-1 2 3 4 5 6 7 86kb~77clones. The nucleotide sequence of theSSrRNA and 5.8S coding regions was deter-mined from both strands. The T. JoetusSSrRNA is 1571 bp long with a G+C contentof 48.5%, and it is presented in a secondarystructure format in Fig. 2. The sequence of thepart of the unit containing the SSrRNA, ITS1,5.8S, and ITS2 regions is available in theGenBank database (M81842). The core fea-tures of the secondary structure are similar tothose established for the Escherichia coli 16SrRNA [16], and form an overall structure thatis common to eukaryotic SSrRNAs. Nucleo-tides for which comparative sequence analysiscan not discern a common structure are left5.8kb4.0kb2.5kb-.,1-- 1.6kbL S 5.8 L SS AE CX RSm A CBSt EH CSt EI II II I II I II II IDI 1kb IFig. 1. (A) Southern blot analysis of the restriction enzyme digested T. Jbetus genomic DNA (1 #g) probed with 32P-labeledtotal RNA. DNA samples from 2 different strains, Crop-1 (lanes 1,3,5, and 7) and UT-1 (2,4,6, and 8), were used. Lanes 1 and2, PvuIl; 3 and 4, Sstl; 5 and 6, Sinai; 7 and 8, XhoI. (B) Northern blot analysis of total RNA (2/~g) probed with 32p-labeledp433. (C) Organization of the rDNA unit. (D) Restriction map of T. Jbetus rDNA unit. E, EcoRl; C, ClaI; X, Xhol; R,EcoRV; Sm, Sma|; A, Accl; S, SaII; B, BstX I; St, Sstl; H, HindlIl.
    • 78unstructured. The only region left unstructuredis between bases 557 and 677. Regions outsidethe core structure are smaller compared withmost other eukaryotic SSrRNAs [9,16].The nucleotide sequences of 5.8S rRNA,ITS l, and ITS2 are shown in Fig. 3. TheU~AUc 1150u u% AC 750 ~ ~u Ao,, •U C Q A GAG = G ~ I I I1 ( I*l CCAAUUA A A UACUCGUUUCUGUuAAuGu CC AUGAGA AGA A GC A I I I I I I I ¢ I I 4 " I • I ( ( I UG • l r i p 1 4 ~ i • r ° l ~ i a iAA U • (k UG C UG Q,AUOG O GGUUAA U C U UGAGAUAGAGACkGUGGC AA G cOGUA CUU UA UCUA UA CG C_ O O G C A GA^ u G oA c~u G.u A........ 6SO 700 A~ . . . . .O U 6 0 C .C ° -- C~u Uu ~,-,.)u .,. u u .,, - 1200G G • A C ~U.Go G A~U CC G A U G.UU U ~ ~ C ~ l a ~ ~ -- CAA~UCAAUGA U mU G * U UA . A C UQ _ CG ~ 800 A-U AUuuuu// UUuO.UU U ~ l C ~ U U uG ~ l C U U, % ~,:,~ ? , "O,c~-oo,U GUCCGUCAAGAc// UCAA o-CCU U 0U % (i A C =c c u_" G i, A UC ,,*d ~ ~.u 12S0A A .GA w~ A--Uc ~ u c / Co A uu ° c A cuu c a% ~ 550 % UA~850 ~ oG ,, ~ ~ ....^: oo ", ..... ~^o°.,~ .~ ~ ~.~uA A cu~ A ~111= • Uu 1000 a%- uC-G ccuAA Gc A G ~C A U CGGCUUG ~ AC~" UUAA u G--C A U GU )AAc^ ~A C G u u Gu, A G C (;U ~--C U Gc ~ ~G60o .,% ,u ,c ~ • ~ ~:c u o,/,% ~, , ~.~ -^ ^ ,,,,:%cSUc . _uGU ~ b¢¢.0 A U~uu C -- O ~UU ~U A CU/ U • G c" A~G ~ Co^CUU(kCu * ~ CC A* a G AA U C U ~ U UCc O.U u G C-e GA uc CAA U U 0U GGAACUACGACC G G~C AG ~/G G--C U • II c A--uAC A GuU % CAUU / uo a~c u A 0 ~.U uc u~.~.cC // C U--A A U CA UC~aA (~ x AAc//AG AGUC -- G U ~JA AAG Q~t~ nGUk ii GCA°o,~°, ~..... ~ "t ....... ~o,,.... o,o,co,,=.,oo:• ~C ~U CC~ AccGUcc/! II II. IIIIIli u C¢ G uC ~-u u A ~, u ac UOU UUCCC(IU U g uA AG--C uU., ~ U u UO A C •u u =u, ~u ~c c c c u ~ .uUO~uuo u u A~ x A A C U A ~ " UU I)lll ~11~ xAC U a 0 A uUuCCAAC ACAU u ~:A Uc C A A C //~U A U°°°~ ,, ,o°° "o, %° ooUUCGGA OGUO G ~ u C A UAACOQUAG~U G O A A~Ue,(,. Ill" G: G A .......... A ~cA CU UGA~=CUc cCC~U u uCZA =uu=cc=ucc, 1~-00 AA• ,,~° Y, ~-o 1c-(~a - c~.50 c - o •C-Gc= 350 A-u uA ~I c-~U--AUg,uu~u/ ~ u ~ G - CAGAUA~cGAc A GU~ U GUUC GO A--UU--AU CGA Ill*it I* II=I A G*UA--U300 .¢c aucC=C==A c= UCA AA GcA C--G• ;."~t% "= ,° ~ ,~uooG,o,.~9, <,~,o°,o" °-°¢ouu - A< 2 , - u • 1500u-AC~ o x CA Ac" u-^u c • c O-C A--UA U c-(; u.ou CuP o-c I00 oc-a,OGu O-c o • A• C U-A=~, ~,; u.o, %-~"• ,>.uo .250 ,-u~ c:~u~x,d ~ o-c uu a~ A-U c c ~cU c--o (L.~Uc-oc c 1~0A -- u uu~GC A e 0 uUuo AU CA CAGUA~$$$ , II.II I~ Ill AUCCC A ~uACAUA OUGGUCuUo-c au¢U-AO.uAa ~"U{iA--U200 a -u,o.u0-¢(;-¢A--UAC -- 0.~A~A oUUFig. 2. TI Joetus SSrRNA primaryand secondarystructure.The higherorderstructureof the SSrRNA was determinedbycomparativemethodsas described[16].
    • 79T. foetus rDNA: ITS1, 5.8S, and ITS21 TTCGTTAATA ATTACAAACA TATTTTTTTA ATTTCTATAA CTATTTATAC51 AAAATTAAAC ACATAATCTA AAAAATTTAG ACCTTAGGCA ATGGATGTCT101 TGGCTTCTTA CACGATGAAG AACGTTGCAT AATGCGATAA GCGGCTGGAT151 TAGCTTTCTT TGCGACAAGT TCGATCTTTG AATGCACATT GCGCGCCGTT201 TTAGCTTGCT AGAACACGCA TATATGTTAC AGTAACCCAT ATTAATTTAA251 TACCAAATTC TCTTTTTAAG CAAAAGAGCG AAAAACAAAT ATGTATTAAC301 AAFig. 3. The nucleotide sequence of the ITSI, 5.8S and ITS2of T. foetus rRNA. DNA sequence analysis of both DNAstrands was performed using Sequenase 2.0 (US Biochem-icals) or modified T7 DNA polymerase (Pharmacia) by thedideoxy nucleotide chain termination method as described[12]. Primers used were either universal [15] or specificoligonucleotide primers synthesized on an Applied Biosys-terns 380B DNA synthesizer at the ICBR DNA synthesisCore at the University of Florida. Some sequences weregenerated from progressively deleted clones obtained bytreatment with exonuclease III/S1 nuclease using Erase-a-base kit (Promega). Sequence analysis was performed withvarious University of Wisconsin Genetics Computer Groupsequence analysis programs [17].boundaries of each of these regions wereextrapolated from comparisons with eukaryo-tic SSrRNA sequences in the GenBank andEMBL data bases. The length of the 5.8SrRNA is 159 bp with a G-C content of 44%.The ITSs are very short at 80 and 64 bp,respectively.The 5-end of the mature SSrRNA of T.foetus was confirmed by the primer extensionanalysis of the total RNA using reversetranscriptase (Fig. 4, panel 2). The Fig. 4,panel 1 shows the dideoxy DNA sequenceanalysis of p433 using the same primer. Thesequence at the 5 end of the molecule startswith ATGAA. Since the primer used forsequencing is a reverse primer, the 5 end ofthe coding strand sequence of the SSrRNA is 5UACUU .... 3. In addition, the 5 end sequencedetermined by homology comparison of thesequence, using GAP or BESTFIT programsof the University of Wisconsin GeneticsComputer Group [17], to other eukaryoticSSrRNAs present in GenBank and EMBLdatabases was consistent with this result. Theprimer extension experiment also enabled us todetermine the 5 end of the primary transcript.Several pauses in the 5 upstream region1GATC2GATCAC ......~ "if5OO2502OO175Fig. 4. Primer extension analysis of the T.foetus SSrRNA.The rDNA transcription initiation sites were determined byoligonucleotide-directed primer extension. The 17-merprimer, 5-GCCCGGAGTCAACTTTT, was extended at42°C using avian myeloblastosis virus reverse transcriptasein a dideoxy sequencing reaction [12]. The primer-extendedproduct was analyzed on a 6% polyacrylamide sequencinggel adjacent to the products of a dideoxy chain terminationsequencing reaction performed on cloned rDNA using thesame sequencing primer. Panel 1, dideoxy sequencingreaction of p433. Panel 2, reverse transcriptase extensionof the total rRNA.
    • 80A2000100000.0• i200 400 600 800 ng0.5 1.0 1.5 2.0 ngB78 kb-39 kb-23 kb-1 2 3 49.4 kb6.6 kb IFig. 5. (A) Gene copy number analysis of the T.Jbetus rDNA. Genomic DNA 750,400, 200, and 100 ng) and P433 DNA (1.6,0.8, 0.4, 0.2 and 0.1 ng) was digested with Xho! and copy number analysis was determined by hybridization analysis ofgenomic DNA using nick-translated cloned rDNA (p433) probe. Decreasing amounts of genomic and plasmid DNA digestedwith XhoI were separated on a 0.8% agarose gel, blotted, and hybridized with the labeled p433 probe. The hybridized blot wasscanned in an AMBIS radioanalytic imaging system.., genomic DNA; [5], cloned rDNA. (B) Southern blot analysis ofpartially digested genomic DNA of T. foetus. Samples of T. foetus total nucleic acids containing 1 /zg genomic DNA werepartially digested with different dilutions of Xhol in a 20/A reaction for 10 min. DNA fragments were separated on a 1%agarose gel using a CHEF apparatus [34]. Size standards were a mixture of HindIII-digested 2 DNA and the ), DNAconcatamer Delta 39 (Promega, Madison, WI). The electrophoresis conditions were: 2.5 second pulse time, 200 V, 12°C, 0.5 x32TBE [12], and 16 h run time. Blots were prepared and P-labeled p433 was used as a probe. Lane 1, undigested genomic4 14 l 3 IDNA; lane 2, 6.6 x 10 units XhoI /A ;lane3,2 x 10 units XhoI /d ;lane4,6.6 x 10 unitsXhollA .represent the presence of minor population oftranscripts at various stages of processing.Major pause sites detected were at approx.175, 200 and 250 bp upstream from the matureSSrRNA. These represent processing sites atthe 5-end of the transcript. Very faint bandsseen approx. 500 bp upstream may representthe true transcription initiation site.The copy number of the rDNA unit of T.foetus was calculated as a percentage of thegenome. As shown in Fig. 5A, the 6-kb XhoIrDNA band in 400 ng of the genomic DNA isequivalent to 1.12 ng of the fragment cloned inp433. Since the genome size of T. foetus isabout 2.5 x 10 7 bp [18], these results indicatethat 12 copies of rDNA units are present pergenome. The presence of only 12 copies ofrDNA unit in T. foetus is relatively lowcompared to other eukaryotic organisms,where the rDNA units are usually repeated100-5000 times [2]. However, it was possiblethat we did not detect the presence of anyextrachromosomal elements carrying rDNAsequences since we used a CsTFA isopycnicgradient centrifugation method to isolategenomic DNA. Therefore, we looked for theexistence of extrachromosomal DNA elementscontaining rDNA sequences using prepara-
    • tions of total nucleic acids. Southern blots of apartial XhoI digest of T. foetus total nucleicacids separated on pulsed field gel electrophor-esis were probed with 32p-labeled p433 (Fig.5B). A ladder of about 12 bands at an intervalof 6 kb was found which is consistent with thecopy number analysis. Behavior of the rDNAin this partial digest also suggests that rDNA isarranged as a linear, tandem repeat of 12 unitslocated within the chromosomal DNA. Geno-mic DNA prepared in agarose blocks wereseparated by pulsed field electrophoresis andhybridized with the same probe. Probe hybri-dized as a band at the position of theunresolved intact chromosomal DNA (> 500kb) and as a smear at 25-70 kb, whichpresumably was partially degraded chromoso-mal DNA (data not shown). Extrachromoso-mal DNA was not detected in this experiment.Neither was it detected by the Hirt fractiona-tion method [19] nor by fractionation of T.foetus genomic DNA on a Hoechst dye 33258 -CsC1 gradient, which separates DNA based onG-C content [20].DiscussionThe T. foetus rDNA repeating unit having amolecular length of 6 kb is one of the smallestreported thus far for a eukaryotic organism.The length of the rDNA unit varies widelyamong eukaryotes, ranging from 5.5 kb inGiardia intestinalis [7] to 44 kb in rat [8].Length variation occurs in tandemly repeatedrDNA units both within and between species[2,8,21-23]. This is primarily due to differencesin the length of the intergenic or non-transcribed spacer region, although lengthdifferences in the coding region and intronsmay also contribute. No rDNA restrictionfragment length polymorphism was detectedbetween 2 different isolates used in this study.The sequence analysis showed that the lengthof the SSrRNA is only 1571 bases, one of theshortest of all eukaryotic sequences reportedthus far [9]. Only Vairimorpha necatrix with1244 nucleotides and Giardia lamblia with 1453nucleotides are shorter. Though not compel-81ling as an argument in light of the large size ofthe T. cruzi SSrRNA, the presence of a shortSSrRNA is a feature of several eukaryoticorganisms which appear to have diverged frommost eukaryotic species early in their phylo-genetic history. It has been proposed that all 3organisms, G. lamblia, V. necatrix and arelated trichomonad, Trichomonas vaginalis,have branched off early in the eukaryoticsubtree [24], although the sequence of the T.vaginalis SSrRNA has not been reported. Allof these organisms are parasitic protozoalacking mitochondria. Phylogenetic distanceanalyses based on a partial sequence of thelarge subunit rRNA of T. vaginalis have alsoplaced the trichomonads on a deep branch ofthe tree of eukaryotes [25].At this time there are over 100 eukaryoticSSrRNA sequences known, spanning themajor phylogenetic groups. Comparative ana-lysis of these sequences has defined a corestructure; a primary and secondary structure,by definition, common to all sequences in theeukaryotic data set [9,16] (R.R. Gutell, un-published analysis). This core region containsin it primary and secondary structure elementsfound within all life forms as well as structurescommon only to eukaryotes. This type ofanalysis also reveals those regions that showextensive variation, the so called variableregions. These regions have the characteristicfeatures of varying in size, base composition,with little or no primary structure conservationacross all eukaryotic sequences. However,within these variable regions there are varyingdegrees of primary and secondary structureconservation within local phylogenetic group-ings.The T. foetus SSrRNA primary structure,while reduced in size when compared withother eukaryotic SSrRNA sequences, doesmaintain the major characteristic eukaryoticprimary and secondary structure features seenin the core structure. The smaller size of thisSSrRNA can be attributed entirely to trunca-tions occurring within the eukaryotic variableregions, most notably in the 2 regionsbracketed by T. foetus bases 171 219 and557-678. A more detailed analysis of the
    • 82trichomonad SSrRNA higher-order structureand phylogeny, in relation to other eukaryoticSSrRNA structures is currently in progress.Internal transcribed spacers in T. Joetus arevery short relative to higher eukaryotes. Forexample, in Xenopus laevis rDNA ITS 1 and 2are 557 and 262 bases, respectively [26], incontrast to 80 and 64 bases, respectively, in T.foetus. A view on the evolutionary origin ofITS2 suggests that it has evolved from asegment of the large subunit rRNA ofprokaryotes [27], since prokaryotes processout only ITS1 resulting in the large subunitrRNA sequence containing the equivalent ofthe eukaryotic 5.8S rRNA and ITS2 at its 5end. Eukaryotes studied to date which havediverged early either lack ITS2 and a separate5.8S as in V. necatrix [28], or have very shortITSs, as in G. lamblia (ITSI: 41 bp; ITS2:55bp) [7]. The internal transcribed spacers, ITS1and ITS2 were very A + T-rich, 86% and 81%respectively. This is in contrast to anotheramitochondrial parasitic protozoon, G.larnblia, where ITSs are C-rich sequences(ITSI: 60%, ITS2: 49%) [29].The large subunit rRNA at 2.5 kb is one ofthe shortest reported so far. Even G. lambliaand E. coli large subunit rRNAs with 3011 and2904 nucleotides, respectively, are longer thanthat of T. foetus. In Northern blots of T..foetustotal RNA probed with the rDNA clone, nosmaller rRNA species were detected, but 2larger transcripts were identified at 5.8 kb and4 kb. Since the 5.8-kb band is most likely to bethe primary transcript, this suggests that thenon-transcribed spacer is approximately 200bp long. The structure of the 4-kb transcripthas not been evaluated. At this moment we areuncertain about the sequence of processingcleavages of the primary rRNA transcript in T.foetus. However, the primer extension experi-ment showed that processing sites are locatedapproximately 250, 200 and 175 bp upstreamof the 5 end of the SSrRNA.Copy number analysis of the rDNA unit ofT. foetus as a percentage of genomic DNAshowed the presence of 12 copies. These copiesarranged in tandem were also resolved in apartial restriction enzyme digestion followedby CHEF electrophoresis. Such a tandemarrangement of rDNA units is a feature incommon with most eukaryotes, whereas therelatively small number of rDNA units con-trasts with organisms such as G. lamblia withan estimated approx. 300 copies of its rDNAunit per genome equivalent [30]. Where a lowcopy number has been reported, such as theapicomplexan protozoans, Babesia bigemina[3] and Plasmodium berghei [4] with only 3 and4 copies of rDNA units, respectively, therDNA units are not tandemly arranged. Otherprotozoan organisms such as Tetrahymena[31], Naegleria [32], and Entamoeba [33] havenumerous copies of extrachromosomal DNAelements carrying rDNA sequences. Uponcareful analysis by various fractionationmethods, no extrachromosomal rDNA wasidentified in T. foetus.AcknowledgementsThis research was supported by grants fromthe University of Florida Institute of Food andAgricultural Sciences and the State of FloridaHigh Technology and Industry Council Ap-plied Research Grants Program. We wouldalso like to thank the W.M. Keck Foundationfor their generous support of RNA science onthe Boulder campus. RRG is an associate inthe Evolutionary Biology Program of theCanadian Institute for Advanced Research.References1 Skirrow, S.Z and BonDurant, R.H. (1988) Bovinetrichomoniasis. Vet. Bull. 58, 591 603.2 Long, E.O. and Dawid, I.B. (1980) Repeated genes ineukaryotes. Annu. Rev. Biochem. 49, 727 764.3 Reddy, G.R., Chakrabarti, D., Yowell, C.A. andDame, J.B. (1991) Sequence microheterogeneity of thethree small subunit ribosomal RNA genes of Babesiabigernina: expression in erythrocyte culture. NucleicAcids Res. 19, 3641 -3645.4 Dame, J.B. and McCutchan, T.F. (1983) The fourribosomal DNA units of the malaria parasite Plasmo-dium berghei: identification, restriction map, and copynumber analysis. J. Biol. Chem. 258, 6984 6990.5 Unnasch, T.R. and Wirth, D.F. (1983) The avianmalaria Plasmodium lophurae has a small number of
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