INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Systems for stable transformation of malaria parasites and the targeted integration of exogenous DNA into their genome have recently been developed (22, 23), now allowing a genetic approach to study Plasmodium protein function in vivo. Integration of targeting constructs into the genome of Plasmodium spp. occurs only by homologous recombination. In addition, the stage of the parasite that can be transformed (the forms that replicate in host erythrocytes) is haploid, enabling study of the function of proteins encoded by single-copy genes after single targeting events.

So far, only null mutations have been created in Plasmodium spp.: in the human parasite P. falciparum (4) and the rodent parasite P. berghei (9, 16). Although null mutations are valuable for revealing the basal function of the target protein, a better understanding of protein function is obtained by altering critical residues or discrete regions of the protein. Several strategies may be used for introducing subtle gene modifications by gene targeting. A modified version of the gene can be expressed via autonomously replicating episomes in a parasite line bearing a null mutation in the gene, a strategy that requires two selectable markers (only one is available in the P. berghei transformation system). The modified gene can also be created by a single recombination event at the wild-type (wt) locus, promoted by a targeting plasmid of the insertion or the replacement type. Figure 1a illustrates the use of an insertion plasmid for gene modification. The targeting sequence of the plasmid lacks the 5' end of the gene, ends after its 3' regulatory elements, and contains the mutation to be expressed. When the plasmid is linearized by introduction of a double-strand break (DSB) in the targeting sequence upstream from the mutation, the final locus contains a full-length, expressed copy of the modified gene, followed by a truncated, nonexpressed copy of the gene.

View larger version (9K): [in this window] [in a new window]   FIG. 1.   (a) Introducing a subtle gene modification with an insertion plasmid. The target gene (symbolized by an open box) is flanked by untranslated sequences (thin lines) bearing the promoter (arrow) and 3' signals (circle) necessary for normal gene expression. The targeting construct contains a bacterial plasmid and a resistance cassette (thick lines) and a targeting sequence that starts after the start codon of the gene, stops after its 3' regulatory sequences, and bears a mutation (asterisk). The plasmid is linearized (gap) within the region of homology upstream from the mutation. Plasmid integration at the target locus duplicates the region of homology and places the mutation in the first, full-length, and expressed copy of the gene. (b) DSB repair model for plasmid integration (ends-in recombination). Plasmid DNA strands are shown in thick lines, chromosomal strands are shown in thin lines, and DNA synthesis is indicated by dashed lines. (Line 1) The initial DSB made within the plasmid sequence is enlarged by exonuclease activity to a double-strand gap, and the plasmid ends are processed to 3'-overhanging, single-stranded tails. (Line 2) One 3' tail invades the homologous duplex and primes repair synthesis while producing a D loop that will anneal to the other 3' tail, thus allowing the second round of repair synthesis. (Line 3) After ligation, the recombination intermediate contains two Holliday junctions embracing the region of gap repair that may be resolved independently as a crossover (by cutting the outer strands) or as a noncrossover (by cutting inner strands). Plasmid integration, which requires one crossover and one noncrossover, occurs in 50% of the cases. (Line 4) The integrated structure contains regions of repaired DNA (a) and heteroduplex DNA (b), which may be asymmetric (on only one chromatid), i.e., promoted by the 3'-single-stranded tails, and potentially symmetric (covering the same region of two chromatids) when the Holliday junction branch migrates.

Homologous recombination with insertion plasmids cut in their region of homology (ends-in recombination) has been widely studied in yeast and mammalian cells. Plasmid integration is thought to occur by a DSB-gap repair mechanism (see Fig. 1b). This model in yeast cells evolved from the demonstration that insertion plasmids cut at two restriction sites, so as to liberate an internal segment of the targeting sequence, still integrated at high frequency at the target locus through a process that always repaired the missing segment (11, 13, 14). The two salient features of this model of recombination (19) are the enlargement of the initial DSB to a double-strand gap, which is filled by two rounds of single-strand repair synthesis, and the formation of two regions of heteroduplex DNA (hDNA) flanking the gap, whose potential mismatches may be converted to either of the sequences. In both yeast and mammalian cells, gene conversion tracts associated with plasmid integration may be several kilobases in length (1, 5, 6, 8, 15, 17, 18, 20).

Here, we investigated the recombination of linearized insertion plasmids in P. berghei by analyzing recombination reactions promoted by insertion plasmids that contained either base-pair substitutions, insertions, or deletions in their targeting sequence.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Construction of control integration plasmids. Plasmid pInco consists of (i) the bacterial plasmid pBSKS (~3 kb); (ii) a DHFR-TS mutant, pyrimethamine-resistance gene from P. berghei expressed from its own 5' and 3' untranslated regions (UTR) (~4.5 kb); and (iii) the distal part of the TRAP gene, lacking the first 22 codons, and 1.4 kb portion of downstream UTR (~3 kb). This TRAP targeting sequence was cloned from genomic DNA of TRAP knockout INT parasites (16) after digestion with HpaI and EcoRV, ligation, and rescue of the resulting plasmid pTRAP-3'UTR in Escherichia coli. Plasmid pTRAP-3'UTR was then digested with BamHI and XmnI, and the TRAP-containing fragment was inserted into plasmid pMD205KpnI-HincII digested with NotI, filled in, and further digested with BamHI. The resulting plasmid, pInco, thus possesses a targeting sequence that originates from the same P. berghei strain (NK65) as that used as a recipient in transformation experiments. Plasmid pInCS is a derivative of pInco that was obtained by exchanging the TRAP targeting sequence by the distal part of the CS gene lacking the first 11 codons and followed by ~0.4 kb of downstream UTR (~1.2 kb).

Construction of mutations. Base-pair substitutions and deletions were created by fusing two contiguous (proximal and distal) DNA fragments amplified by PCR from genomic TRAP (Fig. 2a). The antisense primer used to amplify the proximal fragment and the sense primer used to amplify the distal fragment, which introduce the mutation and a restriction site were, respectively, as follows: R2W, 5'-CGCGAAGCTTCTTCCCATTTTCCACAAAGAGC-3' and 5'-CGCGAAGCTTCTGAATGTTCTACTACATGTGACAATG-3' (HindIII site is underlined); R2+, 5'-CGGTGCTGCAGCTGCAATTGCTGTTCCATTGTCACATGTAGTAG-3' and 5'-CGGCTGCAGCAGTATTACATCCTAATTGTGCTGGAG-3' (PstI site is underlined); S, 5'-CGCTTAATTAACAACAATACCCTTTTCATCATCTGC-3' and 5'-GCGTTAATTAATTTTAATAAACATATATATCTAGAGAATT-3' (PacI site is underlined); and L, 5'-CGCTTAATTAACGCTACTTCCTGCTATAAAATTATAACC-3' and 5'-GCGTTAATTAATTTTAATAAACATATATATCTAGAGAATT-3' (PacI site is underlined). All PCR products were cloned into PCRscript vector by using the PCR-Script cloning kit (Stratagene), and the contiguous fragments were ligated into the pCRScript via the restriction site tagging the mutation. The nucleotide sequence of the fragments used to replace their counterpart in plasmid pInco was determined and verified to differ from that of the wt only by the desired mutation.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   (a) Construction of mutations. The R2W and R2+ base-pair substitutions and the S and L base-pair deletions were constructed by fusing two contiguous PCR-amplified fragments. The antisense primer used to amplify the proximal fragment and the sense primer used to amplify the distal fragment introduced the mutation and a restriction site (asterisk). A fragment encompassing the mutation in the reassembled PCR product (SpeI-HincII or HincII-AflII for the base-pair substitutions or the base-pair deletions, respectively) was then used to replace its counterpart in plasmid pInco. (b) Schematic representation of plasmid pInco and its derivatives. Symbols: thick line, bacterial plasmid; hatched box, resistance cassette; open box, TRAP coding sequence; thin line, TRAP 3' untranslated sequence. The mutations as well as the corresponding wt sequence are shown at the left. Base-pair substitutions are underlined, and restriction sites are italicized. In the wt, the numbers indicate the number of intervening base pairs. The name of the plasmid and the restriction site tagging the mutation are indicated at the right. Abbreviations: A, AflII; B, BamHI; H2, HincII; H3, HindIII; Pa, PacI; P, PstI; S, SpeI.

Construction of plasmid pInco derivatives. To generate plasmids pInR2W and pInR2+, the SpeI-HincII internal fragment of the reassembled locus encompassing the respective mutation was used to replace its counterpart in plasmid pInco. To generate plasmids pInS and pInL, the HincII-AflII internal fragment of the reassembled locus encompassing the respective deletion was used to replace its counterpart in plasmid pInco. The 4-bp insertion in the targeting sequence (TSE) of pInco was obtained by filling in the ends of pInco linearized with AflII, creating a PacI site and resulting in plasmid pInAf. The sequences of the mutations and of their wild-type counterparts are shown in Fig. 2b.

Transformation experiments. Approximately 10 to 30 µg of each plasmid linearized by overnight digestion with the appropriate enzyme (New England Biolabs) was electroporated into ~10⁹ P. berghei merozoites, as previously described (10), and injected into young, susceptible Sprague-Dawley rats. Recipient rats with a parasitemia level of >0.5% at 24 h after injection of the electroporated parasites were treated for 4 consecutive days with pyrimethamine (20 mg/kg of body weight), and the resistant parasites emerged at day 8 or 9 postelectroporation in all experiments. Rats were then treated for 2 additional days, and parasite genomic DNA was collected when the parasitemia level was >1%.

Southern hybridization and PCR analysis of resistant parasite populations. Southern blotting was performed with the entire TRAP coding sequence as a probe. The probe was labelled with DIG-ddUTP by random priming, and the chemiluminescence was detected by using CSPD (Boehringer Mannheim). Genomic DNA of parasite populations was prepared as previously described (10). Specific amplification of the first duplicate of a recombinant locus obtained after integration of plasmid pInco or one of its derivatives was performed with primers O1 (5'-GTTGTGCTTTTATTATGCATAAGTGTG-3', a sense primer that hybridizes to a sequence located at the 5' end of the TRAP coding sequence but absent from pInco) and primer T7 (5'-GTAATACGACTCACTATAGGGC-3', an antisense primer that hybridizes to the bacterial plasmid pBSKS). Amplification of the duplicates located downstream from a cassette was performed with primers OH4 (5'-GCGGAATTCTAATGTTCGTTTTTCTTATTTATATAT-3', a sense primer that hybridizes to a sequence located immediately downstream from the stop codon of the DHFR-TS resistance gene) and primer P18 (5'-GCCGAGCTCAACATTCCATCGTTTTTTTTTATCACAC-3', an antisense primer that hybridizes to a sequence located at the 3' end of the TSE of the plasmids).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Rationale of the study. We performed transformation experiments with a series of targeting insertion plasmids (Fig. 2) into P. berghei parasites. The rodent malarial transformation system (P. berghei parasites infecting rodents) allows rapid in vivo selection of recombinant parasites in rat erythrocytes after transformation with constructs containing a mutant DHFR-TS gene that confers resistance to pyrimethamine (22). The target locus was the single-copy TRAP gene, which is not expressed during the erythrocytic stages of the parasite (16), the stages that can be transformed. TRAP null mutants created in P. berghei by using either insertion or replacement targeting plasmids have been shown to replicate with the same efficiency as that of the wt in rat erythrocytes (16). Since modification of the TRAP locus does not affect parasite replication, the proportion of TRAP integrants in the resistant parasite population should indeed reflect the frequency of initial integration events.

Integration of control insertion plasmids. We first investigated the homology requirements for the integration of the insertion plasmid pInco (insertion control) as its cognate locus. As depicted in Fig. 2b, the TSE of plasmid pInco consists of the distal part of the TRAP gene and 1.4 kb of downstream sequence (~3 kb), thus conforming to the pattern schematized in Fig. 1a. Transformation experiments were performed with plasmid pInco linearized at the SpeI site located 250 bp from the 5' end of the TSE. Figure 3a shows the structure of the recombinant locus generated by integration of plasmid pInco into chromosomal TRAP, called the Inco locus. Parasites with an Inco locus, named Inco, are readily recognized by Southern blot hybridization of parasite DNA by using a TRAP probe and the restriction digestions shown in Fig. 3a. Five independent populations of resistant parasites were analyzed by Southern hybridization, which all contained at least 50% of the expected Inco parasites (Table 1). One representative population, Inco/S4, is analyzed in Fig. 3b. This population contained a majority of Inco parasites, which were detected by the 16- and 4-kb fragments generated upon BamHI digestion and the 14-kb fragment generated upon MscI-EcoRI digestion. Population Inco/S4 also contained a minority of parasites with a wt TRAP, as shown by the presence of the 9.5-kb fragment upon BamHI digestion and of the 3.5-kb fragment upon MscI-EcoRI digestion.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Integration of control insertion plasmids. (a) Schematic representations of the wt TRAP locus, plasmid pInco (10.5 kb), and the Inco recombinant locus generated by homologous integration of pInco at the TRAP locus (not drawn to scale); see below for symbols and abbreviations. The predicted size (in kilobases) of restriction fragments generated upon digestion with BamHI, which cuts once in the plasmid backbone, or MscI-EcoRI, which do not cut in the construct, in a wt TRAP or an Inco locus are shown. (b) Southern hybridization with a TRAP probe of wt P. berghei (lanes 1 and 4), population Inco/S4, obtained after transformation with plasmid pInco linearized at the SpeI site (lanes 2 and 5), and population GAP1 obtained after transformation with plasmid pInco cut at the HincII and PstI sites (lanes 3 and 6). (c) Schematic representations of the wt CS locus, plasmid pInCS (8.5 kb), and the recombinant locus generated by homologous integration of pInCS at the CS locus (not drawn to scale). The predicted size of restriction fragments generated upon digestion with PacI, which cuts in the cassette and in the TSE, or with both BamHI and EcoRV, which cuts or does not cut in the construct, respectively, are shown. (d) Southern hybridization with a CS probe of the wt (lanes 1 and 4), and two populations obtained after transformation with pInCS cut in the TSE either at the HincII site (lanes 2 and 5) or at the AflII site (lanes 3 and 6). Symbols: open box, TRAP or CS coding sequence; thin line, TRAP or CS UTRs; thick lines, bacterial plasmid and resistance cassette (not drawn to scale). Abbreviations: A, AflII; B, BamHI; E, EcoRI; E5, EcoRV; H2, HincII; M, MscI; N, NdeI; Pa, PacI; P, PstI; S, SpeI; X, XbaI.

Fate of base-pair substitutions. We then analyzed the fate of base-pair substitutions in the TSE of plasmid pInco during homologous integration. As shown in Fig. 2a, these mutations, named R2W and R2+ and consisting of 5- and 11-bp substitutions, respectively (Fig. 2b), were created by amplification-cloning. The plasmid pInco derivatives pInR2W and pInR2+ which contained the respective substitutions were cut at the SpeI site, located ~450 bp upstream from the mutations, and independently transformed into P. berghei. Most of the resistant populations obtained (Table 1, populations R2+ and R2W/S) contained a high proportion of parasites which had the plasmid integrated into TRAP, suggesting that heterologies in the TSE did not dramatically affect the integration efficiency. The presence of the respective mutations in the recombinant loci was assessed by Southern blot hybridization of parasite DNA digested with both HincII and the restriction enzyme corresponding to the mutation tagging site. The fragments diagnostic for the presence of the mutation in the TRAP integrants were not detected in any of the populations obtained (Table 1). This indicated that both mutations were frequently corrected during recombination initiated by a DSB located ~450 bp upstream.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Fate of base-pair substitutions and insertions during recombination. (a) Recombinant locus generated by homologous integration of plasmid pInR2W at the TRAP locus. The predicted size (in kilobases) of restriction fragments generated upon digestion with BamHI, which cuts once in the bacterial plasmid, or with both HincII, which cuts in the TSE, and HindIII, which cuts in the cassette, are shown. The presence of the HindIII-tagged mutation (asterisk) in the second duplicate is revealed by the cleavage of the 1.6-kb fragment into two smaller fragments. (b) Southern hybridization by using a TRAP probe of populations R2W/S3 and R2W/P4 obtained after transformation with plasmid pInR2W linearized at the SpeI or the PstI site, respectively. The BamHI digestion indicates that both populations contained a majority of integrants, and the HincII-HindIII digestion shows the presence of the additional HindIII site in the second duplicate of virtually all parasites in population R2W/P4 but not population R2W/S3. (c) Recombinant locus generated by homologous integration of plasmid pInAf at the TRAP locus. The predicted size of restriction fragments generated upon digestion with HincII-AflII or PacI are shown. When HincII-AflII was used, the absence of the wt AflII site (asterisk) in the first duplicate is indicated by an additional 10.5-kb fragment, whereas when PacI was used the presence of the PacI-tagged mutation in the first duplicate is indicated by an additional 7-kb fragment. The PCR fragment amplified by primers O1 and T7 is shown below. (d) Southern hybridization with a TRAP probe of an Inco clonal population (lanes 1 and 3) and population Af/P4 (lanes 2 and 4), obtained after transformation of plasmid pInAf linearized at the PstI site. (e) Restriction analysis of the first TRAP duplicates amplified by PCR with primers O1 and T7 (see panel c). The majority of the PCR products amplified from population Af/P4 lack the wt AflII site and contain the PacI-tagged mutation. Symbols and abbreviations are as in Fig. 3.

Fate of a sequence insertion. We then tested the fate of a sequence insertion in the TSE of plasmid pInco during plasmid integration. Four base pairs were inserted in pInco by filling in the overhanging ends of the unique AflII site, a process that destroys this site and creates a PacI site (Fig. 2b). The resulting plasmid, pInAf, was cut with one of the restriction enzymes, PstI, HincII, or SpeI, located at increasing distances from the mutation and was used in independent transformation experiments. The correct integration events were detected in all populations, and integrants with the mutation in the first TRAP copy were obtained with each linear form of the plasmid (Table 1). Importantly, the 4-bp insertion was reproducibly maintained in the recombinant locus even when the initiating DSB was located only 600 bp away (populations Af/P-1 to Af/P5). Conversely, the mutation was also corrected in a proportion of integrants in population Af/S1, indicating that the mutation may also be corrected during an integration process initiated ~2 kb away.

Fate of deletions. We then tested the fate of deletions in the TSE of plasmid pInco during recombination. The plasmid pInco derivatives pInS and pInL were constructed which lacked 36 and 105 bp at the 3' end of the TRAP coding sequence, respectively (Fig. 2b). Transformation experiments were performed with each of these plasmids linearized with SpeI, which cuts ~1,400 bp from the deletions. All seven resulting populations contained at least 50% of recombinants having one and sometimes more copies of the plasmid integrated into TRAP. In three populations, the corresponding deletion was present in the first duplicate in more than 50% of the recombinant parasites (Table 1). One such population, L3, is analyzed in Fig. 5a to c. As depicted in Fig. 5a and shown by Southern hybridization in Fig. 5b, this population contained wt-like parasites and parasites with one copy of the plasmid integrated into TRAP. The replacement of the 9.5-kb fragment by a 1.8-kb fragment upon MscI-PacI digestion demonstrated that the PacI-tagged deletion was present in the first TRAP duplicate in virtually all integrants. This was confirmed by restriction analysis of PCR products amplified with primers O1 and T7, which specifically amplify the first TRAP duplicates (Fig. 5c). All products amplified from population L3 indeed possessed the PacI site tagging the deletion, but not the PstI site located in the deleted segment. The ~100-bp deletion was also visible upon comigration of the XbaI-digested products amplified from an Inco clonal population and population L3. Finally, we also introduced a ~600-bp deletion (HincII-PstI internal fragment of TRAP) in the first TRAP copy by using a similarly deleted pInco derivative linearized at the SpeI site located ~800 bp upstream (data not shown). These results indicate that large sequence heterologies may be maintained during homologous recombination.

View larger version (30K): [in this window] [in a new window]   FIG. 5.   Fate of deletions during recombination. (a) Recombinant locus generated by homologous integration of plasmid pInL at the TRAP locus. The predicted size (in kilobases) of restriction fragments generated upon digestion with both MscI and EcoRI, which do not cut in the construct, and MscI and PacI, which cut in the cassette, are shown. The presence of the PacI-tagged deletion (asterisk) in the first duplicate is revealed by the cleavage of the 9.5-kb fragment into a 1.8-kb fragment. (b) Southern hybridization with a TRAP probe of the wt, an Inco clonal population, and population L3 obtained after transformation with plasmid pInL linearized at the SpeI site. Population L3, which contains a minority of wt-like parasites, mainly consists of integrants having the deletion in the first duplicate. (c) Restriction analysis of the first TRAP duplicates amplified by PCR with primers O1 and T7. The restriction map of the PCR products amplified from a wt (Inco) or deleted (L3) duplicate are shown. All PCR products amplified from population L3 possess the deletion. (d) Recombinant locus generated by homologous integration of more than one copy of plasmid pInL at the TRAP locus. The predicted size of the BamHI- and PacI-generated fragments are shown, as well the PacI-generated fragments if the PacI-tagged deletion (asterisks) is present in each of the duplicates. (e) Southern hybridization with a TRAP probe of clones L4A and L4B. Note that digestion with BamHI, which cuts only once in the plasmid backbone, liberates a fragment of the size of the plasmid (11 kb) only when multiple plasmids are integrated. (f) Restriction analysis of PCR products amplified by using primers OH4 and P18 that amplify all duplicates located downstream from a cassette. PacI digestion of the PCR products demonstrates the presence of the PacI-tagged deletion in all duplicates of clone L4B but not clone L4A. , Uncut DNA. Other symbols and abbreviations are as described in Fig. 3.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We studied recombination events in P. berghei between the TRAP genomic locus and several types of mutant alleles borne by insertion plasmids. As shown in previous studies (10, 22), insertion plasmids in their uncut, supercoiled forms do not integrate at a detectable level into the genome of P. berghei and plasmid integration is stimulated by introduction of a DSB in the TSE of the plasmid. The recombination reactions promoted here by linearized insertion plasmids were in agreement with the DSB repair model of recombination (19). The model (Fig. 1b) predicts that a mutation located in the region of homology of the plasmid may be corrected during recombination if it falls either within the gap, which is repaired by using wt sequence as a template, or within the flanking hDNA, which may be converted to the wt sequence by the mismatch correction system. As verified in many studies (1, 17, 18), the frequency of correction declines with increasing distance between the DSB and the mutation, and gene conversion tracts can extend from the initiation site in both directions during the same recombination event.

Gene conversion associated with plasmid integration in mitotically dividing yeast cells is thought to occur both via gap expansion and rectification of mismatches in hDNA. The typical length of asymmetric hDNA adjacent to the gap, involving the single-stranded tails, has been estimated to be several hundred base pairs in the yeast plasmid transformation system (12), whereas potential symmetric hDNA, generated by branch migration of the Holliday junctions, may extend up to 4 kb from the DSB (8, 15). Double-strand gap repair has also been demonstrated in mammalian cells (2, 20). Studies have reported that mutations located ~1 kb from the DSB in an insertion plasmid had only a 25% probability of faithful transfer to the mammalian genome (5, 20), whereas a mutation located ~4 kb from the DSB had a 95% probability of being maintained in the recombinant locus (5).

The results we obtained here in P. berghei can be summarized as follows. (i) Mutations located close to the DSB (up to ~0.45 kb) were frequently corrected during plasmid integration. Such a disparity in favor of correction events may suggest that these mutations were degraded via gap extension. However, the fact that insertion plasmids cut 250 bp from one end of the TSE integrated efficiently (at two different genomic loci) suggests that the gap that may form prior to integration and presumably extends in both directions (12, 17) is frequently smaller than 250 bp. The frequent correction of proximal mutations may thus mainly be due to a mismatch correction system that strongly favors the chromosomal strand in the hDNA located close to the DSB. Such differential mismatch repair bias at different distances from the initial site has been reported in yeast cells (6). (ii) Mutations located as far as 1.5 to 2 kb from the DSB could also be corrected during plasmid integration, such as in population Af/S1. This indicates that hDNA can propagate through most of the TSE before resolution of the recombination intermediate and that mismatch repair may still favor the chromosomal strand in hDNA located far from the DSB. (iii) Conversely, a 105-bp deletion located ~1,500 bp from the DSB could be transferred from the plasmid to the chromosome (clone L4B, see Fig. 5). This implies not only that hDNA may form between two strands bearing large heterologies but also that mismatches in hDNA may be repaired to the plasmid sequence (which presumably occurred in both chromatids of symmetric hDNA in clone L4B). (iv) All mutations (substitutions, insertions, and deletions) located as close as 600 bp and farther from the DSB were nonetheless frequently maintained during recombination. With mutations located between 600 bp and 1 kb from the DSB, 4 of 8 experiments yielded populations with 50% or more of the mutated recombinants, whereas with mutations located more than 1 kb from the DSB, 6 of 11 experiments yielded such populations. These results suggest that the hDNA either frequently stops before it reaches ~600 bp and/or it may progress farther but is then associated with a mismatch correction system that no longer favors the chromosomal strand.

The main feature of the plasmid transformation system for homologous recombination in yeast or mammalian cells or P. berghei is that plasmid integration is dramatically increased by introduction of a DSB in the TSE of the plasmid. Surprisingly, this appears not to be the case in P. falciparum, in which linearized insertion plasmids are not more proficient for homologous recombination than their circular counterparts (3, 23). However, the transformation procedures differ substantially between the two plasmodial species, not only in the selection methods (in vitro for P. falciparum and in vivo for P. berghei) but also in the parasites transformed (intra-erythrocytic blood stages for P. falciparum or extracellular merozoites for P. berghei). Although some difference in processing linear DNA may exist between the two species, it seems more likely that linear DNA may be more efficiently degraded during P. falciparum transformation.

In P. berghei, the ends-in strategy should be widely applicable to analyze the structure-function relationships of relevant malarial proteins. An ends-in strategy has several advantages over alternative methods for introducing subtle gene modifications. Because the modified gene is expressed at the original locus, it is likely that it is subjected to the same chromosomal effects as the wt gene. In addition, the recombinant locus (Fig. 1a) contains uninterrupted sequences located upstream from the target gene (up to the first duplicate) as well as downstream (starting from the second duplicate). Thus, neighboring genes should be minimally affected by plasmid integration. In contrast, the recombinant locus generated at the target locus with a replacement plasmid (ends-out recombination) contains the foreign DNA upstream (or downstream) from the regulatory sequences of the modified gene, which may disrupt a closely linked locus or affect its expression. Finally, the locus generated by ends-in recombination is stable for numerous generations in the absence of drug pressure, including in mosquito stages of the parasite (16). Methods relying on expression of the modified gene from nonintegrated episomes are limited by the high variability in the copy number of the episomes, as well as by the need to apply selective pressure for maintaining the plasmid in replicating parasites.

We have shown here the reproducible generation of ends-in integrants that had maintained various mutations located at least 500 bp from the DSB. These mutant parasites could in most cases (14 of 19 experiments) be cloned by limiting dilution from first-generation populations obtained at ~days 10 to 11 postelectroporation. With only 250 bp for the short arm of homology sufficient for crossing-over and the short length (~500 bp) of gene conversion tracts, it should be possible to express any modification in a P. berghei gene of 1.5 kb by a single-step ends-in strategy.