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• W1970738156 abstract "The rapid developments in the molecular genetics of Toxoplasma gondii have far reaching implications in treatment and vaccination strategies for this as well as closely related pathogens such as Plasmodium. Although stable transformation of this parasite through homologous and illegitimate genomic integration has provided many of the tools necessary for genetic analysis, subsequent manipulations of the DNA have proven laborious. This report describes the selection and subsequent characterization of a Toxoplasma sequence that permits the episomal maintenance of bacterial plasmids in this parasite. This sequence was isolated from the Toxoplasma genome through selection for episomal stability of a pUC19-based library in the absence of a selectable marker. A 500-base pair fragment was determined to possess the stabilization activity. Transformations of Toxoplasma using vectors possessing this fragment, referred to as EMS (episomal maintenance sequence), demonstrated an elevated stable transformation frequency compared with the vector alone. Mutants deficient in hypoxanthine-xanthine-guanine phosphoribosyltransferase activity were used as a test to see if this gene could be selected from a genomic library using a vector containing the EMS. The success of this test demonstrates the utility of EMS-containing vectors in complementation strategies and the ability of such constructs bearing large fragments of the Toxoplasma genome to be maintained episomally. The rapid developments in the molecular genetics of Toxoplasma gondii have far reaching implications in treatment and vaccination strategies for this as well as closely related pathogens such as Plasmodium. Although stable transformation of this parasite through homologous and illegitimate genomic integration has provided many of the tools necessary for genetic analysis, subsequent manipulations of the DNA have proven laborious. This report describes the selection and subsequent characterization of a Toxoplasma sequence that permits the episomal maintenance of bacterial plasmids in this parasite. This sequence was isolated from the Toxoplasma genome through selection for episomal stability of a pUC19-based library in the absence of a selectable marker. A 500-base pair fragment was determined to possess the stabilization activity. Transformations of Toxoplasma using vectors possessing this fragment, referred to as EMS (episomal maintenance sequence), demonstrated an elevated stable transformation frequency compared with the vector alone. Mutants deficient in hypoxanthine-xanthine-guanine phosphoribosyltransferase activity were used as a test to see if this gene could be selected from a genomic library using a vector containing the EMS. The success of this test demonstrates the utility of EMS-containing vectors in complementation strategies and the ability of such constructs bearing large fragments of the Toxoplasma genome to be maintained episomally. Toxoplasma gondii is an obligate intracellular protozoan of the phylum Apicomplexa which includesPlasmodium, Eimeria, and other medically and agriculturally important pathogens. Although many of the structural and biochemical attributes of these pathogens are conserved, Toxoplasma is unusual within the phylum in several characteristics that are amenable to molecular genetic manipulations (1Roos D.S. Donald R.G.K. Morrissette N.S. Moulton A.L.C. Russell D.G. Methods in Cell Biology. 45. Academic Press, Inc., San Diego1995: 28-63Google Scholar, 2Boothroyd J.C. Kim K. Sibley D. Soldati D. Boothroyd J.C. Komuniecki R. Molecular Approaches to Parasitology. 12. John Wiley and Sons, Inc., New York1995: 211-225Google Scholar). For example, the lack of host cell specificity and rapid replication cycle allows one to quickly and inexpensively propagate this organism to high numbers in vitro. Since the parasite has a haploid genome in the asexual part of its life cycle, molecular genetic manipulations are not complicated by allelic copies, and generation of loss-of-function mutations can be done easily by a variety of methods (3Pfefferkorn E.R. Pfefferkorn L.C. Exp. Parasitol. 1976; 39: 365-376Crossref PubMed Scopus (156) Google Scholar, 4Pfefferkorn E.R. Exp. Parasitol. 1977; 44: 26-35Crossref Scopus (57) Google Scholar, 5Pfefferkorn E.R. Pfefferkorn L.C. J. Parasitol. 1979; 65: 364-370Crossref PubMed Scopus (30) Google Scholar). In addition, homologous and illegitimate recombination for the stable transformation of constructs into the genome of this parasite have been widely used in the analysis and complementation of different genetic loci (6Donald R.G.K. Roos D.S. Mol. Biochem. Parasitol. 1994; 63: 243-253Crossref PubMed Scopus (75) Google Scholar, 7Donald R.G. Roos D.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11703-11707Crossref PubMed Scopus (280) Google Scholar, 8Kim K. Boothroyd J.C. Clin. Res. 1993; 41: 209AGoogle Scholar, 9Sibley L.D. Messina M. Niesman I.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5508-5512Crossref PubMed Scopus (104) Google Scholar). Although genomic integration is conducive for a variety of genetic manipulations (e.g. increased stability of the transforming DNA, definitive replacement of a gene, etc.), some procedures are restricted by the nature of this transformation event. Using an episomal vector to stably transform a strain would bypass many of these obstacles and would allow easy recovery and/or removal of a given construct. The intrinsic instability associated with episomal DNAs allows definitive evidence for the association of a given phenotype to the DNA under examination using a molecular form of Koch's postulates. Since the vector would be independent of the parasite's genome, analysis of the activity attributed to the transformed DNA would simply require either isolating the episome to re-transform the parental strain or selecting against the episome using a negative selectable marker. In addition, as the episome does not directly interact with the genome, the probability of inducing a mutation as a result of integration is diminished. To obtain sequences that would allow independent replication and stabilization of episomes in Toxoplasma, we searched through the parasite's genome to recover sequences that demonstrate these attributes. Although the majority of the procedures previously used to isolate autonomous replicating sequences (ARS) 1The abbreviations used are: ARS, autonomous replicating sequences; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyltransferase; EMS, episomal maintenance sequence; kb, kilobase pair(s); bp, base pair(s); DHFR, dihydrofolate reductase; MPA, mycophenolic acid; cfu(s), colony-forming unit(s); EM, episomal maintenance.1The abbreviations used are: ARS, autonomous replicating sequences; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyltransferase; EMS, episomal maintenance sequence; kb, kilobase pair(s); bp, base pair(s); DHFR, dihydrofolate reductase; MPA, mycophenolic acid; cfu(s), colony-forming unit(s); EM, episomal maintenance. from eukaryotic organisms have utilized selectable markers (10Kelly J.M. Ward H.M. Miles M.A. Kendall G. Nucleic Acids Res. 1992; 20: 3963-3969Crossref PubMed Scopus (193) Google Scholar, 11Godberg J. Salazar N. Oren R. Mirelman D. Nucleic Acids Res. 1990; 18: 5515-5519Crossref PubMed Scopus (14) Google Scholar, 12Stinchcomb D.T. Thomas M. Kelly J. Selker E. Davis R. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4559-4563Crossref PubMed Scopus (248) Google Scholar), we have chosen to select for sequences that stabilize episomal copies in the absence of drug pressure to achieve a strong selection for sequences providing a highly efficient mode of replication and/or faithful segregation. The selection for these sequences and characterization of a 500-bp fragment that possesses this activity are described in this report as is a test of an engineered shuttle vector in a complementation experiment. The RH strain (13Sabin A.B. J. Am. Med. Assoc. 1941; 116: 801-807Crossref Scopus (297) Google Scholar) and mutants generated from this strain were grown in the tachyzoite stage for all experiments presented. The parasites were propagatedin vitro by serial passage in monolayers of human foreskin fibroblasts as described (14Roos D.S. J. Biol. Chem. 1993; 268: 6269-6280Abstract Full Text PDF PubMed Google Scholar). The HXGPRT mutants in this strain consist of the RHΔHXGPRT knock-out strain (1Roos D.S. Donald R.G.K. Morrissette N.S. Moulton A.L.C. Russell D.G. Methods in Cell Biology. 45. Academic Press, Inc., San Diego1995: 28-63Google Scholar, 15Pfefferkorn E.R. Borotz S.E. Exp. Parasitol. 1994; 79: 374-382Crossref PubMed Scopus (50) Google Scholar) and the TXR-2 point mutant. TXR-2 was isolated from a population of RH mutagenized with 150 μg/ml N-nitroso-N-ethylurea (Sigma) for 60 min, syringe-released, and re-plated on a fresh monolayer for selection using 6-thioxanthine at 400 μg/ml (Sigma). After 2 weeks of continuous selection, clones were isolated that were incapable of growth under the selective pressure of mycophenolic acid confirming their status as deficient in HXGPRT activity (see below). The selection of parasites transformed with the chloramphenicol acetyltransferase was performed using 20 μmchloramphenicol (Sigma) as described previously (8Kim K. Boothroyd J.C. Clin. Res. 1993; 41: 209AGoogle Scholar). Strains deficient in HXGPRT activity were selected for the transformation of a construct bearing a form of the HXGPRT gene using 50 μg/ml mycophenolic acid (MPA) and 50 μg/ml xanthine (Sigma). Individual plaques of MPA-resistant clones were picked from monolayers overlaid with selective medium containing 0.9% Bacto-agar as described previously (1Roos D.S. Donald R.G.K. Morrissette N.S. Moulton A.L.C. Russell D.G. Methods in Cell Biology. 45. Academic Press, Inc., San Diego1995: 28-63Google Scholar). All constructs used in the transformation of Toxoplasma in this report use the pANA vector backbone. This vector was generated by inserting a 90-bp sequence containing a centralNotI site flanked by two AscI sites in pUC19 at the AflIII site. Modifications of this vector include the following: 1) pANA-0.5, adding the 500-bpEcoRI/KpnI fragment of pEM1 (constituting the episomal maintenance sequence (EMS)) in the respective sites of the multicloning site (MCS); 2) pANAE, adding a blunted version of the 500-bp EMS in a blunted NotI site (outside the MCS); 3) pCANA, adding a blunted BamHI/HindIII tubulin-driven chloramphenicol acetyltransferase cassette of pT/230 (16Soldati D. Boothroyd J.C. Mol. Cell. Biol. 1995; 15: 87-93Crossref PubMed Scopus (110) Google Scholar) in a SspI site; 4) pCANA-1.9, the pCANA vector with the 1.9-kb KpnI fragment of pEM1 in the MCS; 5) pHANA, adding a DHFR-driven HXGPRT cassette of pmini-HXGPRT (17Donald R.G.K. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) in a SspI site; 6) pHANA-0.5, the pHANA vector with the 500-bp EMS in the uniqueEcoRI and KpnI sites of the MCS. The blunted restriction sites were generated using the Klenow fragment of Escherichia coli DNA polymerase I, and all ligations were performed using T4 DNA ligase by standard techniques (18Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. J. Wiley and Sons, Inc., Boston1995Google Scholar). The pACYC184 vector (New England Biolabs) was used as a control for DNA extractions and subsequent transformations of E. coli. A genomic library of the PDS strain of Toxoplasma gondii (a clonal isolate of ME49 (19Ware P.L. Kasper L.H. Infect. Immun. 1987; 55: 778-783Crossref PubMed Google Scholar)) was used to select sequences bearing episome-stabilizing activity in the RH strain. This library was generated in the BamHI site of pANA using a Sau3AI partial digest of genomic DNA from which fragments 4–8 kb in size were gel-purified (Geneclean) using standard procedures (18Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 1. J. Wiley and Sons, Inc., Boston1995Google Scholar). Two additional libraries of RH genomic DNA were constructed similarly in pANA and pANAE for the isolation of the HXGPRT gene. The complexity of each of the three libraries was found to be 1–5 × 105 independent recombinants with >90% containing Toxoplasma genomic DNA as determined by the lack of β-galactosidase activity and size analysis of random clones. T. gondii (RH strain) were transformed with 50 μg of the PDS genomic library as described previously (20Soldati D. Boothroyd J.C. Science. 1993; 260: 349-352Crossref PubMed Scopus (287) Google Scholar). In the first two rounds of selection for episomal maintenance, the transformed parasites were passaged twice in human foreskin fibroblasts monolayers and recovered to isolate genomic and episomal DNAs using the TELT procedure described below. The DH12 strain of E. coli was electroporated with the isolated DNA and plated on ampicillin plates to select for bacteria transformed with recovered episomal copies. Plasmid was extracted from the population of ampicillin-resistant bacteria for subsequent transformation into RH for additional rounds of selection. The population of plasmids carried through four passages of RH in the third round were re-electroporated into RH for an additional five passages in the fourth and final round of selection. Following recovery of episomal DNA from this population and transformation of E. coli, plasmids from 20 ampicillin-resistant bacterial clones were digested with RsaI and examined for the enrichment of certain library fragments as determined by their restriction pattern. Five distinct constructs were chosen based on the criterion of being represented at least twice among the bacterial clones examined. T. gondii DNA used in the preparation of genomic libraries was isolated from a freshly lysed culture in a single T-175 flask. Recovered parasites were syringe-released using a 27-gauge needle and separated from host cell debris using a 3-μm nucleopore membrane (Costar). Parasites were pelleted, washed, and resuspended in a lysis solution containing 120 mm NaCl, 10 mmEDTA, 25 mm Tris (pH 8.0), 1% Sarkosyl, and 0.1 mg/ml of RNase A. After a 30-min incubation at 37 °C, proteinase K (1 mg/ml) was added for an additional incubation overnight at 55 °C. The genomic DNA was twice extracted with phenol:chloroform, ethanol-precipitated, and resuspended in 10 mm Tris (pH 8.0), 1 mm EDTA. DNA of Toxoplasma used for the quantitation and analysis of episomal forms was extracted from parasites freshly lysed from T-25 monolayers using a modified version of the TELT extraction procedure (21Medina-Acosta E. Cross G. Mol. Biochem. Parasitol. 1993; 59: 327-329Crossref PubMed Scopus (231) Google Scholar). Parasites were syringe-released using a 27-gauge needle, pelleted, and lysed using a solution of 50 mm Tris (pH 8.0), 62.5 mm EDTA, 2.5 m LiCl, and 4% Triton X-100. Genomic and episomal DNA was extracted twice using phenol:chloroform, ethanol-precipitated, and resuspended in 10 mm Tris (pH 8.0), 1 mm EDTA. To take into account the differences in recovery of episomal DNA and subsequent transformation of E. coli, equal amounts (∼1 ng) of the tetracycline-resistant plasmid pACYC184 were added to each pellet of cells prior to DNA extraction (pACYC184 possesses a plasmid p15 origin of replication that is compatible with the ColE1-type origin on the pANA-based vectors). Using this, the number of ampicillin-resistant colonies could be normalized to the number of tetracycline-resistant colonies obtained from the same electroporation. The pACYC184 “spike” also allowed the transformation efficiency of the extracted plasmids to be determined which in turn enabled the actual number of plasmid molecules per parasite to be estimated. To determine the actual number of transformation-competent episomes per parasite, this normalized figure was then multiplied by the difference between the number of TetR obtained from a known amount of the pACYC184 plasmid and the number recovered from the cell pellet after the DNA extraction procedure. For Southern blot analysis, genomic and episomal DNAs were digested with either EcoRI or PvuII, subjected to electrophoresis, and transferred to a nylon membrane for hybridization to random-primed radiolabeled probes corresponding to either the pANA vector or a 700-bp BamHI/NdeI fragment of the HXGPRT gene. Phosphorimaging analysis of hybridized membranes was done using the Storm 860 PhosphorImager (Molecular Dynamics) and quantitated using ImageQuant software (Molecular Dynamics). AToxoplasma genomic DNA library was generated from aSau3AI partial digest of the PDS strain with an average insert size of 6 kb using the pUC19-based vector pANA. The PDS genomic DNA was used to generate the library as this strain has only recently been derived from an oocyst, whereas the RH strain has been in continuous lab passage for over 50 years. Thus, PDS is expected to have undergone fewer genetic alterations (i.e. deletions) under the selection for in vitro growth. The RH strain of Toxoplasma was electroporated with 50 μg of this library and passaged two times through confluent monolayers of human foreskin fibroblasts. After the second passage, genomic and episomal DNAs were extracted and electroporated into the DH12 strain of E. colifor selection of the episomes bearing the ampicillin resistance marker of pANA. Plasmids isolated from populations of ampicillin-resistant bacteria were re-transformed into the RH strain for an additional three rounds of selection. From the resulting population, 20 ampicillin-resistant E. coli colonies were picked and their plasmids analyzed by restriction endonuclease digestion usingPvuII. Of these 20, 5 clones were chosen that showed distinct digestion patterns and appeared to be represented at least twice among those analyzed (data not shown). As our ultimate goal was to generate a shuttle vector forToxoplasma that could be efficiently retained in episomal form even in the absence of selection, the five isolated plasmids were analyzed for this property. After three passages of RH transformed with the isolated constructs and a pANA control, all five clones were at least 100-fold more efficient in maintaining the constructs as episomes in RH in the absence of selection when compared with the parental vector alone (see Fig. 1). Since the constructs of the library had an average insert size of 6 kb, a control construct in the same vector carrying a ∼5.8-kb XmaI fragment from a cosmid clone of SAG1 was similarly tested and demonstrated no stabilizing activity (data not shown). One of the five clones, named pEM1 (episomal maintenance), was arbitrarily chosen for further analysis. As the ∼6.8-kb size of the pEM1 genomic insert is excessively large for use as a stability element in episomal vectors, we sought to isolate a smaller sequence from this clone that possessed the same EM activity. As a first step in identifying the critical region in this insert, a restriction map of pEM1 was generated, and overlapping fragments of the insert ranging from 0.75 to 4.2 kb were isolated and cloned into pHANA (the modified form of pANA carrying hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) driven by upstream and downstream non-coding sequences of dihydrofolate reductase (DHFR) (17Donald R.G.K. Carter D. Ullman B. Roos D.S. J. Biol. Chem. 1996; 271: 14010-14019Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar)). A constant molar amount of each construct (5–10 μg each) was electroporated into a strain of RH deleted in HXGPRT open reading frame (RHΔHXGPRT (1Roos D.S. Donald R.G.K. Morrissette N.S. Moulton A.L.C. Russell D.G. Methods in Cell Biology. 45. Academic Press, Inc., San Diego1995: 28-63Google Scholar, 15Pfefferkorn E.R. Borotz S.E. Exp. Parasitol. 1994; 79: 374-382Crossref PubMed Scopus (50) Google Scholar)) and grown in the presence of 50 μg/ml mycophenolic acid (MPA) and xanthine to select for HXGPRT expression. Only parasites transformed with the construct carrying a 1.9-kbKpnI fragment (pHANA-1.9) were able to survive after the second passage under drug pressure. DNA isolated from the third passage of these parasites revealed that at least part of the population carried the marker episomally (data not shown, but see below). It has been previously suggested that sequences in the dihydrofolate reductase gene of Toxoplasma are able to elevate the stable transformation frequency (6Donald R.G.K. Roos D.S. Mol. Biochem. Parasitol. 1994; 63: 243-253Crossref PubMed Scopus (75) Google Scholar, 7Donald R.G. Roos D.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11703-11707Crossref PubMed Scopus (280) Google Scholar). Although there are only short regions of the DHFR locus in the pHANA vector and the aforementioned activity was not observed using other fragments of the pEM1 genomic insert, we chose to use a construct bearing a tubulin-driven chloramphenicol acetyltransferase gene (pCANA) to examine the relative transformation frequency in a different context. Plasmids carrying the 1.9-kb fragment (pCANA-1.9) were compared with a control construct with a 2.0-kbPstI fragment of the parental EM1 clone that did not possess episomal maintenance activity (pCANA-2.0). Transformed parasites were passage three times in chloramphenicol and plated for plaque formation to compare the number of surviving parasites between the control and EMS-bearing constructs. Since chloramphenicol kinetics are such that parasite death is only detected during the third passage, approximately 16–20 generations would have occurred before the chloramphenicol acetyltransferase marker exerts its positive effects. The pCANA-1.9 fragment was determined to elevate the transformation frequency by ∼53-fold when compared with the pCANA-2.0 size control (data not shown). Although this indicates that the 1.9-kb fragment elevates the transformation frequency, more definitive evidence will be presented below demonstrating that the augmentation of transformation is a result of stabilizing the constructs in an episomal form, thus eliminating the requirement for the less frequent genomic integration event. As the 2.0-kb PstI fragment, which does not possess stabilizing activity, overlaps 1.4 kb of the 1.9-kb KpnI fragment, the remaining 500 bp of this DNA were examined for episomal maintenance activity. This region was cut out of the 1.9-kb parental sequence using flanking EcoRI and KpnI sites (KpnI derived from the multicloning site of pANA) and ligated into the pANA plasmid (pANA-0.5). The pANA and pANA-0.5 vectors were electroporated into RH and examined for EM activity in the absence of selective pressure as described above using pACYC184 to normalize for variations in DNA extractions and electroporations (see “Experimental Procedures”). Fig.2 A shows that the number of colony-forming units/parasite arising from DNA of the pANA-0.5 transformants is maintained at a higher level than the number recovered from those electroporated with the pANA vector. As there is no detectable difference in activity when pANA-0.5 was compared with a pANA-based vector carrying the parental 1.9-kb fragment (data not shown), we conclude that this 500-bp sequence confers the episomal stability accredited to pEM1. Although the pANA vector carrying the EMS did not stabilize the vector at the same levels as seen with the parental pEM1 clone (see Figs. 1 and 2 A), we suggest that this variance is a result of the ∼6.8-kb difference in size between the two vectors as discussed below. The complete sequence for this 500-bp EMS has been determined (GenBank accession #AF009625). No significant sequence homology was detected when compared with the available data bases, and there were no conserved motifs apparent in comparisons with known autonomous replicating sequences and centromeres. Although Southern blot data indicate that the 500-bp sequence is found in a single copy in the genome (data not shown), this assay may not detect a small conserved motif that could be more abundant. The other four constructs isolated (pEM2 to pEM5) were not used to probe genomic DNA because the critical regions responsible for EM activity have not been identified to generate specific probes. Since the insert size of these constructs is ≥7 kb, of which it is suspected only a small portion actually confers episomal maintenance, finding multiple bands would be meaningless as one or more may result from the hybridization of repetitive elements carried in the genomic insert (22Ossorio P.N. Sibley L.D. Boothroyd J.C. J. Mol. Biol. 1991; 222: 525-536Crossref PubMed Scopus (57) Google Scholar). Southern blots of PvuII-digested DNA from all five of the vectors isolated carrying EM activity (pEM1-pEM5) were individually probed with the 500-bp EMS or the pEM2-pEM5 constructs to determine if there are sequence similarities between the individual EMSs. No cross-hybridization between any of the five probes (outside of the common pUC19 backbone) was detectable. Thus there is no substantial similarity in the sequences found within the library inserts. Until the critical regions in the other four plasmids (pEM2-pEM5) are identified and sequenced, we cannot exclude that they share some small, critical motif. Regardless, for our purposes, the 500-bp EMS identified here is of convenient size and possesses the activity desired. To demonstrate active replication of the episomes, the methylation status of DNA recovered from Toxoplasma was examined using thedam methylase-sensitive restriction enzymes DpnI (only cuts DNA methylated by dam) and DpnII (will not cut dam-methylated DNA). Inasmuch as this methylase activity is not found in eukaryotic organisms, and the DNA used in all parasite transformations was from the dam(+) DH12 strain of E. coli, the loss of methylation (i.e. resistance to DpnI) would verify that the DNA was replicated by the parasite. Genomic and episomal DNAs isolated from the second and third passage from the experiment described above (data shown in Fig.2 A) were digested with either DpnI orDpnII and electroporated into E. coli. Fig.2 B shows that as early as the second passage, there is a clear difference in the number of colony-forming units arising from the digests between the EMS-bearing pANA-0.5 and parental vector. The activity of the enzymes in each reaction was confirmed by showing that the dam-methylated pACYC184 spike was virtually eliminated by the DpnI digest (≤0.01% number of cfus obtained without digest) and not affected by the DpnII digest (data not shown). These data demonstrate the active replication of unselected episomes carrying the 500-bp EMS. Interestingly, the DpnI resistance detected in the vector lacking the EMS (see Fig. 2 B) suggests that a small number of these plasmids was replicated by the parasite. Although this appears to be a significant alteration of the methylation status when compared with the pACYC184 control, there is no way of controlling for differences in accessibility of the enzyme between the vectors recovered from the parasites and the pACYC184 spike. The different methylation profiles of pANA and pANA-0.5 at the second and third passage indicate that the difference in the number of replicated (DpnI-resistant) episomes is due either to the specific replication of pANA-0.5 episomes (ARS activity) or “nonspecific” replication of the constructs with the selective maintenance of those bearing the EMS (centromeric activity). Nonspecific replication refers to an unidentified ARS-like activity already present in the vectors analyzed without the requirement for Toxoplasma sequences. This form of episomal replication was observed in Leishmania(22Ossorio P.N. Sibley L.D. Boothroyd J.C. J. Mol. Biol. 1991; 222: 525-536Crossref PubMed Scopus (57) Google Scholar) and may be responsible for the prolonged transient transformation observed in Toxoplasma (see below). However, the relative numbers of enzyme-resistant vectors and the comparison of the total number of episomes between the EMS ± vectors (see Fig.2 B) suggest that the constructs of the pANA-0.5 transformation are being actively converted from aDpnII-resistant phenotype to one that is resistant toDpnI. This would support the hypothesis that the activity ascribed to the EMS is one of elevating the replication competency of the plasmid in Toxoplasma (i.e. ARS-like activity). The 500-bp EMS was subcloned into pHANA (pHANA-0.5) for a more quantitative analysis of transformation frequency under MPA selection in place of the slow kinetics of chloramphenicol activity. A total of 2 × 106 plaque-forming units of the RHΔHXGPRT strain were electroporated with 10 μg of pHANA or pHANA-0.5, and 5 × 103 or 1 × 103 were immediately plaqued with or without MPA, respectively. An additional sample (5 × 104) of this electroporated population was grown under MPA selection to propagate the HXGPRT-transformed parasites as a passage flask for subsequent plaque assays. After sufficient time for plaque development, the parasites plated for the plaque assay were fixed and stained for analysis, while those grown in the passage flask (which had not yet completely lysed) were syringe-released, counted, and plated for a second round of plaque assay (± MPA) and passage in MPA(+) media. This procedure was repeated for five passages. After the second passage, the population corresponding to the pHANA transformation was not able to form plaques in drug-free medium, whereas the pHANA-0.5 population maintained a consistent percentage of viability throughout the selection (see Fig. 3). The inability to form plaques in drug-free medium after growth in MPA demonstrates that the plaques of the second passage were not viable at the time of harvesting" @default.
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• W1970738156 date "1998-02-01" @default.
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• W1970738156 title "Development of a Stable Episomal Shuttle Vector for Toxoplasma gondii" @default.
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