Volume 15 Number 5 1987 Nucleic Acids Research Karyotype comparison between P.chabaudi and P.falcqxinsm: analysis of a P.chabaudi cDNA Karyotype colnparison between P. chabal and P.falcyparum: analysis of a P. ca cDNA contning sequences highly repetitive in P.falkiparum Gordon Langsley*, Lise Sibilli, Denise Mattei, Pierre Falanga and Odile Mercereau-Puijalon Unite de Parasitologie Experimentale, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, France Received December 29, 1986; Revised and Accepted February 6, 1987 Abstract The molecular karyotypes of P.chabaudi and P.falciparum have been compared by pulse field gradient electrophoresis. P.chabaudi has 3 extra chromosomes in the 750 - 2000 Kb range although the overall number appears to be 14 as is the case for P.falciparum. The chromosomal location of the rRNA genes has been determined for P.chabaudi together with that of a 24 Kd antigen gene. The corresponding cDNA 443 may code for a protein unusually rich in tyrosine and contains sequences highly repetitive in P.falciparum. Introduction Part of the genome of Plasmodium falciparum has recently been separated into its constituent chromosomes (1) (2). These studies demonstrated that the chromosomes of P.falciparum are relatively small (850-5000 kbp) and highly polymorphic in size. The size polymorphism can be generated by deletions of repetitive DNA (1) or of coding sequences, which in one case resulted in a change from the knob+ to knob- phenotype (3, 4). More recently the pulse field gradient (PFG) separation technique has been improved and the genome fractionated into 10 bands (5). The number of chromosomes now visualized is approaching the number of kinetocores seen by electron microscopy (6, 7). Since the molecular karyotype of P.falciparum appears to be a stable characteristic in vitro (1,2,3,5), the chromosomal rearrangements leading to size polymorphism probably occur during sexual reproduction. This has been demonstrated for chromosome 4 following a defined genetic cross between 2 P.falciparum clones (8). P. chabaudi, a parasite of rodents has long been used as a model to study malaria parasites (9,27). One reason for this is the close evolutionary relatedness between the rodent malarias and the human malaria parasite P.falciparum (10). Previously P.chabaudi has been used for genetic analysis due to the difficulty in performing crosses with P.falciparum. Genetic diversity, inheritance of drug resistance and the genome haploidy of Plasmodia were also established using rodent parasites (11). As a first step towards an understanding of the genetic control of chromosome size polymorphism through karyotype analysis of defined P.chabaudi crosses (Langsley, C I RL Press Limited, Oxford, England. Nucleic Acids Research Volume 15 Number 5 1987 2203
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Volume 15 Number 5 1987 Nucleic Acids ResearchKaryotype comparison between P.chabaudi and P.falcqxinsm: analysis of a P.chabaudi cDNA
Karyotype colnparison between P.chabal and P.falcyparum: analysis of a P.ca cDNAcontning sequences highly repetitive in P.falkiparum
Gordon Langsley*, Lise Sibilli, Denise Mattei, Pierre Falanga and Odile Mercereau-Puijalon
Unite de Parasitologie Experimentale, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, France
Received December 29, 1986; Revised and Accepted February 6, 1987
AbstractThe molecular karyotypes of P.chabaudi and P.falciparum have
been compared by pulse field gradient electrophoresis.P.chabaudi has 3 extra chromosomes in the 750 - 2000 Kb rangealthough the overall number appears to be 14 as is the case forP.falciparum. The chromosomal location of the rRNA genes hasbeen determined for P.chabaudi together with that of a 24 Kdantigen gene. The corresponding cDNA 443 may code for a proteinunusually rich in tyrosine and contains sequences highlyrepetitive in P.falciparum.
IntroductionPart of the genome of Plasmodium falciparum has recently
been separated into its constituent chromosomes (1) (2). Thesestudies demonstrated that the chromosomes of P.falciparum arerelatively small (850-5000 kbp) and highly polymorphic in size.The size polymorphism can be generated by deletions ofrepetitive DNA (1) or of coding sequences, which in one caseresulted in a change from the knob+ to knob- phenotype (3, 4).More recently the pulse field gradient (PFG) separationtechnique has been improved and the genome fractionated into 10bands (5). The number of chromosomes now visualized isapproaching the number of kinetocores seen by electronmicroscopy (6, 7). Since the molecular karyotype of P.falciparumappears to be a stable characteristic in vitro (1,2,3,5), thechromosomal rearrangements leading to size polymorphism probablyoccur during sexual reproduction. This has been demonstrated forchromosome 4 following a defined genetic cross between 2P.falciparum clones (8).
P. chabaudi, a parasite of rodents has long been used as amodel to study malaria parasites (9,27). One reason for this isthe close evolutionary relatedness between the rodent malariasand the human malaria parasite P.falciparum (10). PreviouslyP.chabaudi has been used for genetic analysis due to thedifficulty in performing crosses with P.falciparum. Geneticdiversity, inheritance of drug resistance and the genomehaploidy of Plasmodia were also established using rodentparasites (11). As a first step towards an understanding of thegenetic control of chromosome size polymorphism throughkaryotype analysis of defined P.chabaudi crosses (Langsley,
C I RL Press Limited, Oxford, England.
Nucleic Acids ResearchVolume 15 Number 5 1987
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Shakey and Walliker, in preparation), we decided to compare thekaryotypes of P.chabaudi and P.falciparum. We demonstrate here,that like P.falciparum the chromosomes of P.chabaudi can beseparated by PFG. We show that P.chabaudi has 3 extra smallchromosomes although the overall number is probably the same asfor P.falciparum. In a search for sequences homologous to bothgenomes we have identified a P.chabaudi cDNA which appears to behighly repetitive in P.falciparum. The derived cDNA sequence didnot indicate the reason for the cross hybridisation.
MATERIAL AND METHODSParasite strains and chromosomal blocks
The Tak 9.96 clone of P. falciparum (12) and the F clone ofP. chabaudi (13) were used for all PFG. gels. The blocks weremade from infected blood at 10% paresitemia as described in (1).For P. chabaudi samples were taken at 30% parasitemia from micepreviously irradiated (600 rads/8 min.) to reduce the number ofwhite blood cells and then diluted. The P.chabaudi AJ blockswere a kind gift of A. Shakey and D. Walliker. Genomic DNA fromP.falciparum Palo Alto FUP Uganda strain (14) was prepared as in(21).Pulse field gradient L OFAGE q and transfers
A gel apparatus identical to that described by Carle andOlson (15) was used at 250 v and 0.5X TRIS/borate/EDTA. The gels(1% agarose) were run for 20 hr. before staining in ethidiumbromide. The pulse was varied from 80-100 seconds and thecurrent when varied was done so by changing the buffer depth.After photography the gels were treated for 15 minutes in O.3MHCI before transfer (16) to hybond-N (Amersham).Hybridisation probes
Telomere probe. The P. berqhei telomer DNA sequence used wasthat described by Ponzi et al, (17).
rRNA genes. The P. falciparum rRNA genes used were thosedescribed by Langsley et al, (18).
cDNA 443. A P. chabaudi cDNA coding for a 24Kd antigen andwhich cross-hybridised with P. falciparum cDNA (19).
cDNA 451 A P. chabaudi cDNA encoding a 27 Kd antigenimmunologically cross-reactive with P.falciparum and weaklypositive with P.falciparum cDNA (19).
DNA seguence of cDNA 443The P. chabaudi DNA insert was excised from PBR322 by PstI
purified and then religated. The circularized insert was thencut by either Sau3AI, isI, Sspi or DraI, cloned into t1.,3 phageand sequenced according to the Amersham protocol for S-dATPand gradient gels.
RESULTSKaryotype profiles of P.chabaudi and P.falciparum
The PFG apparatus used here employed 2 heterogeneouselectric fields which results in the parasite chromosomesmigrating in a straight line and not diagonally as previouslydescribed (3; 4; 5). The separations obtained under differingconditions are shown in Fig.l. An 80 second pulse (Fig IA)separated the P.falciparum genome into only 4 chromosomes(marked with arrows), the remaining larger chromosomes migrated
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A BPfPcC
__~~~~~~~~~~~~~~~~- 7
3 D4
Pf Pc Pt Pc
.4-
.~5.444Jt, 3 0- 4-2
4.;>4
Figure 1 Pulse field gradient gel electrophoresisof P.falciparum (Pf) and P.chabaudi (Pc). The two genomes wereseparated into their constituent chromosomes at an 80 secondpulse (A), 90 second (B) and 100 second pulse (D). In (C) an 80second pulse was used and the current reduced from 130 mAmps to110 mAmps. Increasing the pulse improved the separation of thelarger chromosomes but reduced the degree of separation of thesmaller ones. The chromosomes are numbered from the bottom andshown arrowed in each case are the first 4 chromosomes of Pf andthe first 7 of Pc.
in a large, highly fluorescing band. P.chabaudi however, possesextra smaller chromosomes that were separated into 5 bands ofwhich the first and fourth contain at least 2 chromosomes(arrowed and see below). Again, the remainder of the genomemigrated as a large highly fluorescing upper band. A 90 secondpulse improved the separation of the higher molecular weightchromosomes (Fig. 1B). The amount of DNA, migrating near theslot was higher for P.falciparum than for P.chabaudi, indicatingthe presence of more large chromosomes. Fig. 1D shows theseparation at a 100 second pulse. The quantity of P.falciparumDNA in this block was slightly lower. At this relatively longpulse time the separation of the large chromosomes is furtherimproved, but the definition is poor probably due to shearing ofthe high molecular weight DNA. A similar profile was obtainedusing an 80 second pulse at 110 mAmp (Fig.1C). These conditionsagain resulted in an improved separation of the largerchromosomes but lead to a condensation of the smaller ones.
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Fi qure 2 P.falciparum (Pf) andP.chabaudi (Pc)chromosomes hybridised with telomere sequences. The degree ofhybridisation indicates the chromosome number. In Pc (right handpanel). The first and fourth bands and the slot hybridise more
strongly indicating the presence of more than one chromosome. Atthe 100 second pulse used 11 chromosomes are separated in bothgenomes and are shown arrowed. The fainter hybridisation withthe larger chromosomes is probably due to some DNA loss due toshearing and to poor transfer of this high molecular weight DNA.
Chromosome number estimated by. hybridisation with telomere probeIn Fig. 1 it can be seen under the different conditions
used the 1st and 4th P.chabaudi bands fluoreced more intenselyindicating the presence of another chromosome. This point wasfurther demonstrated when the gel shown in Fig. iD was transfer-red and hybridised with a probe containing P.berghei telomeresequences (Fig. 2). The first and fourth bands hybridised twiceas strongly as the second and third. Hence each band contains 2chromosomes (marked 1 + 2 and 5 + 6). Eleven chromosomes in allcan be identified and given that the slot also hybridised aboutthr,ee times as strongly, the total number of chromosomes isprobably 14. A similar number of P. falciparum chromosomes wasalso identified with the P.berghei telomere sequences. Thedifference being that the P.falciparum genome is composed ofmore large chromosomes. A chromosome number of 14 is inagreement with the electron microscopy data which shows thepresence of 14 kinetocores during sexual division (7).Chromosomal location of rRNA genes and cDNA 443
Since the chromosomal location of the P.falciparum RNAgenes was known (1) and these sequences have been shown tohybridize to P.chabaudi (20), the chromosomal location wasassigned for P.chabaudi (Fig.3, left hand panel). Shown forcomparison is the hybridisation withP.falciparum chromosomesseparated on the same PFG gel. The rRNA transcription units in
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rRNA cDNA443
Pc Pf Pf Pc Pc Pf Pc Pf
P d slot slot ??9 6
8
il 1 *
Fiq 3 Chromosomal mapping of rRNA genes and cDNA 443.The rRNA sequences (left hand panel) hybridised to differentchromosomes in the two genomes. The relevent chromosome beingnumbered and given for both P.chabaudi (Pc) and P.falciparum(Pf). The cDNA 443 hybridised specifically to chromosome 1 inPc. and to all chromosomes in Pf. Note the smear observed at topof gel for rRNA genes is probably due to shearing of highmolecular weight DNA.
P.chabaudi are located on chromosomes numbered 5, 8, 9 and slot.The location on chromosome 5 of the 5/6 doublet was confirmed byhybridisation with the P.chabaudi clone AJ where the twochromosomes separate independantly (data not shown).
The P.chabaudi cDNA clone 443 codes for a 24 Kd antigen andis a unique sequence which hybridises to chromosome 1. Thesignal observed at the slot in the left P.chabaudi track (Fig.3right hand panel) is due to some material being trapped as it isabsent in the second P.chabaudi slot. This P.chabaudi codingsequence hybridised in a highly repeptitive fashion to allP.falciparum chromosomes (Fig. 3 r.h.p.). The repeated elementsare probably present in many P.falciparum strains, as onSouthern blots of restricted genomic DNA they gave a similarrepetitive pattern with the P.falciparum Palo Alto strain whilstbeing unique in the P.chabaudi genome (Fig 4A). The repetitivecharacteristic appears specific for cDNA 443 as P.chabaudi cDNA451 isolated at the same time was negative with P.falciparum(Fig. 4b).DNA sequence of cDNA 443
In an attempt to identify the nucleotides responsible forthe cross-hybridisation with P.falciparum the entire sequence ofclone 443 was determined (Fig.5). As expected cDNA clone 443contains only one large open reading frame which terminates with
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_~C
Olink-
,~~~~~~~~~~~~~~~~~~~~~~~~~~0
Fi 4 Geuiomic DNA of P.chabaudi and_falciarumhybridised with cDNA 443 and cDNA 451. cDNA 443 is a uniquesequence in the genome of P.chabaudi (Fig 4A track 1, Rsi,track 2, Dral) whil-st being highly repetitive in the genome ofP.falciparum (Fig 4A, track 3, RsI, track 4, Sau3A, track 5,
RsI). Shown also less exposed are Fig 4A tracks 3, 4 and 5. Thesame transfer hybridised with cDNA 451 (Fig 4B), which does notcross hybridise with P.falciparum. Size markers are pBR322 cutwith EcoRI, PsI, BamHI and AvaI and the filter was hybridisedin 4xSSC and washed in 2xSSC at 650C.
two stop codons (TAG, TAA). These are fol1lowed by 225 bp of 3'untranslated sequence ending with the poly A tail. This togetherwith the stop codons and the putative poly-adenylation sequenceare underlined. The 5' end of the 24 Kd antigen gene is notcontained within cDNA clone 443. Like other Plasmodia with A+Trich genomes (21, 22, 10) P.chabaudi cDNA 443 has a high A+Tcontent of 80% and a biased codon usage. Eight of the tyrosinesare coded by TAT and only 2 by TAC. Since the coding specificityof cDNA 443 was established by hybrid selection (19) anotherreading frame other than the one presented here may be used in
v 9i- _ wviv.
DiscussionThe genome of P.chabaudi like that of P.falcioarum is
composed of relatively small chromosomes that can be sizeseparated by PFG. Although the total number of chromosomes isprobably the same in both species the proportion of smallerchromosome was found to be higher for P.chabaudi than forP.falciDarum. There being 7 as opposed to 4 in the 750 - 2000 kbrange. The profiles obtained were dependent on the pulse used,
Figure 5 DNA sequence of P.chabaudi cDNA 443. Thenucleotides are deoxyadenosine (A), deoxycytidine (C),deoxyguanosine (G) and deoxythymidine (T). The correspondingamino acid is shown below the DNA sequence and the number ofbase pairs (bp) above. The putative stop and poly-adenylationsignals together with the poly-A tail are shown underlined. ThecDNA is 338 bp long of which 225 bp are 3 prime untranslatedsequence. The 113 bp of open reading frame encode 37 amino acidsof which 10 are tyrosines. The sequence is 81% rich in A and T.
at shorter pulses the size differences in the smallerchromosomes were more apparent. Long pulses improved theseparation of the larger DNA molecules but did result in someDNA shearing at the voltages used.
Hybridisation with the P.berqhei telomere sequences gave anestimate of chromosome number and also showed that in P.chabaudiclone F chromosomes 5 and 6 migrate as a doublet. In all otherP.chabaudi clones examined under these pulse conditions thefirst band was also a doublet containing 2 chromosomes (G.Langsley unpublished). The cross hybridisation of the P.bercheitelomere sequences to the chromosomes of P.chabaudi andP.falciparum confirms the preliminary report of Pace et al (23)and implies that all Plasmodia chromosomes may have similartelomeres.
The location of the rRNA genes on chromosomes 5, 8, 9 inP.chabaudi compared to 1, 5, 6 in P.falciparum raises thequestion of whether these are functionnaly equivalentchromosomes of different size or whether the transcription unitsare located on different chromosomes in the two species. The
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demonstration of chromosome size polymorphism in P.falciparumcomplicates an interpretation based just on size and morechromosomal markers that hybridise to the 2 species are neededbefore one can identify equivalent chromosomes. Furthermore,chromosomal comparisons between polymorphic strains or speciesof Plasmodia would be greatly aided with the establishment ofreference strains, for example Tak 9.96. for P.falciparum.
Finally the P.chabaudi cDNA clone 443 which codes for a 24Kd antigen has been mapped to chromosome 1 in P.chabaudi. ThiscDNA cross-hybridised to every P.falciparum chromosomeindicating that it contains sequences highly repetitive in theP.falciparum genome. The derived DNA sequence revealed no motiveresponsible for the repetitive hybridisation. It is nothomologous to a highly frequent P.falciparum element, the rep 2021bp repeat (24) either by computer analysis or by hybridisation(M. Goman pers. comm.). The hybridisation pattern observed(Fig.4A) was similar to that noted for a repetitive sequencedescribed by Enea (25). The two clones may therefore containthe same repetitive element(s). It seems unlikely however, giventhe unusual distribution of G+C rich restriction sites presentin the repetitive sequence detailed by Enea (25). A computersearch for homology (60% level) with cDNA 443 identified a shortsequence in the cap-oxil region of yeast mitochondrial DNA (26).The biological significance, if any, of this homology is unknownat this time as is the reason why a P.chabaudi coding sequenceshould contain elements highly repeptitive in P.falciparum.
ACKNOWLEDGMENTSWe would like to thank Luiz Pereira da Silva for his
support, David Walliker and Andrew Sharkey for the P.chabaudi AJchromosomal blocks and M. Goman for the hybridisation of cDNA443 with the 21 base pair rep 20 repeat. We thank G. Prensierand C. Slomiany for comunication of results prior to publicationand C. Frontali for the gift of the P.berghei telomeresequences. We also thank FrXddrique L6cuiller for typing themanuscript. D.M. was supported by a Pasteur-Weizmann fellowship.The work was funded by the French Ministry of Industry andResearch (grant nO.0.06.84.4276) and by Institut PasteurProduction.
*To whom correspondence should be addressed
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