Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii

ELSEVIER Molecular and Biochemical Parasitology 74 (1995) 55-63

MOLECULAR

EkEMICAL PARASITOLOGY

Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii

Michael Black”, Frank SeebeTa, Dominique John C. Boothroyd*”

Soldatib, Kami Kim”,

aDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, 94305-5402. USA

bZMBH Zentrum fur Molekulare Biologic. Universitat Heidelberg. 69120 Heidelberg, Germany ‘Department of Medicine, Division of Infeclious Diseases, Albert Einstein College of Medicine, Bronx, NY 30461, USA

Received 4 May 1995; revision received 28 August 1995; accepted 28 August 1995

Abstract

This report describes the use of restriction enzyme-mediated integration (REMI) to increase the transformation frequency and allow co-transfection of several unselected constructs under the selection of a single selectable marker. We found that while BarnHI (the enzyme used to originally demonstrate REMI (Schiestl, R.H. and Petes, T.D. (1991) Inte- gration of DNA fragments by illegitimate recombination in Succharotnyces cerevisiae. Proc. Natl. Acad. Sci. USA 88, 7585-7589) increased the number of transformants by 2-5-fold over the control without added enzyme, NorI proved to be a further 29-46-times more effective in enhancing stable transformation. This simple technique was used in the transformation of three non-selective markers (two modified membrane proteins and fi-galactosidase) with a selectable construct expressing chloramphenicol acetyltransferase. Following chloramphenicol selection, four out of ten independent transformants stably acquired all four constructs with at least two expressing all four genes at the protein level. These results demonstrate that REMI may be used in the efficient stable transformation and co-transfection of this and perhaps other protozoan parasites.

Keywork Restriction enzyme-mediated integration (REMI); NorI; BarnHI; Co-transfection; Transformation

1. IntrOfIuctioo

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. This parasite

is increasing in significance because of its role as the causative agent of toxoplasmic encephalitis in

Abbreviations: bp, base pairs; CAT, chloramphenicol acetyltransferase; UTR, untranslated region; REMI, restriction enzyme-mediated integration

* Corresponding author, Tel.: +I 415 7237984; Fax: +I 415 7236853.

AIDS patients. In addition, Toxoplasma is par- ticularly amenable to investigation of intracellular

parasitism due to an unusually broad range of host cells and the recent development of tools for molecular genetic analysis [2,3]. Such analyses, especially when using genomic libraries for com-

plementation, require an efficient method of transformation to obtain the necessary number of independent transformants. As there are no published self-replicating vectors for Toxoplasma, stable transformation of this parasite is dependent

0166-6851/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-6851(95)02483-T

56 M. Black et al. /Molecular and Biochemical Parasitology 74 (1995) 55-63

on the less efficient process of genomic integration. While exceptionally high frequencies of stable transformation of T. gondii have recently been obtained using a selectable dihydrofolate reductase-thymidylate synthase marker (DHFR- TS, [4]), other markers have not demonstrated this efficiency [5,6]. Since DHFR-TS selection results in the production of strains resistant to pyri- methamine (one of the leading drugs in treatment for toxoplasmosis), we sought out methods of elevating the rate of transformation using safer selectable markers. Additionally, the small number of selectable markers known to work in T. gondii thus far necessarily limits the number of genetic manipulations possible. Hence, we were interested in developing protocols that would also allow each selectable marker to be used to introduce multiple engineered genes. Restriction enzyme-mediated integration (REMI) is a technique that was first used to increase the rate of transformation of linearized DNA in the yeast Saccharomyces cerevisiae [l]. This procedure involved simply adding the restriction enzyme BamHI to an electroporation of a linearized DNA fragment possessing BumHI ends. REM1 was also found to elevate the frequency of transformants in Dictyostelium as long as the enzyme used in the electroporation was complementary to the ends of the linearized DNA [7]. Southern blot analysis from both groups indicated that >70% of these transformants resulted from integration events into BamHI sites of the host genome. The resulting hypothesis for the activity of REM1 was a simple ligation-repair model where the genomic DNA is cut by the introduced restriction enzyme and ligated to the complementary ends of the electroporated DNA.

In this report, we describe the influence of REM1 on the transformation of Toxoplasma

gondii and its utilization in the co-transfection of multiple plasmids. We find that REM1 can dramatically increase the transformation efficiency but by a mechanism other than the ligation-repair model.

2. Materials and methods

2.1. Growth and selection of parasites Tachyzoites of the RH strain [8] were used in all

experiments presented. The parasites were pro-

pagated in vitro by serial passage on monolayers of human foreskin libroblasts as described [9]. Two clones of the B-mutant of strain PLK [lo] of T. gondii (a strain that does not express detectable ~30) that have stably integrated copies of either the pSAG-tm or pSAG-set were used as controls in Southern and Western blots.

The selection of stable transformants was performed using chloramphenicol selection of pT/230 or pT/230* (CAT) recombinants as described previously [5]. Following three passages in human foreskin fibroblasts (2-3 days/passage) under 20 PM chloramphenicol selection, the cultures were diluted (l/16 and l/1600) to obtain distinct plaques in 100 mm tissue culture treated dishes (Falcon) containing a confluent monolayer of HFF [2]. The infection was allowed to proceed for 16 h before the medium was replaced by medium containing 0.9% Bacto agar as previously described [2]. After 5-6 days, individual plaques were picked with plugged Pasteur pipettes and dispensed into 24- well plates for propagation. Plaques were scored in the transformation efIiciency experiments (Fig. 2) by staining plates with either Giemsa stain or crystal violet after 5-6 days of growth [2].

2.2. Constructs The distinct attributes of the four constructs

used in both the transformation efficiency and co- transfection experiments are schematically represented in Fig. 1. pSAG-see was generated by removing 90 bp from the 3 ‘-end of the SAG1 (also called p30 [l 11) open reading frame (ORF) that includes the putative glycophosphatidyl inositol (GPI)-anchor addition signal (Soldati and Boothroyd, unpublished). pSAG-tm was generated by replacing this 3 ‘-region with a sequence encoding a trans-membrane region of human CD46 (Seeber and Boothroyd, unpublished). The pT/230* construct differs from its parental plasmid (pT/230) by the loss of the unique Not1 restriction site. This site was destroyed by digesting the construct with NotI, filling in the recessed termini using the Klenow fragment of E.

coli DNA polymerase I, and re-ligating the blunted ends by standard techniques [12].

2.3. Transformation with REMI Parasites used in the transformation efftciency

hi. Black et al. I Molecular and Biochemical Parasitology 74 (1995) 55-63 51

experiments were transformed with 20 rg of linearized or circular constructs as described in Fig. 2. The linearized constructs were digested with the indicated enzyme and phenol extracted to eliminate residual enzymatic activity (circular forms were treated similarly as a control measure). After ethanol precipitation, the DNA was resuspended in the same cytomix used for the transfection of T. gondii as previously described [13]. In REM1 transfections, 100 units of either BumHI or Not1 (New England Biolabs) were added to the electroporation cuvette containing the parasites and DNA immediately prior to electroporation from stock concentrations of 10 000 and 20 000 U/ml, respectively. Heat inactivation of the enzymes was accomplished using a 20 min in- cubation at 75°C.

2.4. /3-Galactosidase and CAT activity /3-Galactosidase activity was assayed in cell

lysates generated through lysing - 2 x 10’ parasites in 100 mM Hepes (pH 8.0), 1 mM MgS04. 7H20, 1% Triton X-100, and 5 mM DTT at 50°C for 1 h. Chlorophenol red-&D- galactopyranoside is the calorimetric indicator that was used to detect enzymatic activity by spec- trophotometry as previously described [ 14,151. CAT activity was assayed on lysates prepared from 2 x 10’ parasites lysed by three rounds of freeze-thawing in 0.25 M Tris-HCl (pH 7.8). The enzymatic activity of chloramphenicol acetyltransferase was determined using tritiated acetyl-CoA in a phase partition assay as previously described WI.

2.5. Western blot Total lysates were prepared from the ten co-

transfection clones and the two B-mutant controls and subjected to SDS/PAGE. The separated proteins were transferred to nitrocellulose membranes and probed with a monoclonal antibody to p30 (DG52 [ 111). A secondary peroxidase-conjugated goat anti-mouse antibody was used for visualiza- tion by ECL fluorescent detection reagents (Amersham).

2.6. Genomic DNA analysis Genomic DNA was isolated from T. gondii from

a freshly lysed T-175 monolayer. After the para-

sites were serially syringed using 20, 23 and 25 gauge needles, they were separated from host cell debris by filtration through a 3-pm nucleopore membrane (Costar). Extracellular parasites were pelleted, washed, and resuspended in a lysis solu- tion containing 120 mM NaCl, 10 mM EDTA, 25 mM Tris (pH 8.0), 1% sarkosyl, and 0.1 mg/ml of RNase A. The lysate was incubated 30 min at 37°C before adding Proteinase K (1 mg/ml) and in- cubating an additional 2 h at 55°C. The genomic DNA was phenol extracted, ethanol precipitated, and resuspended in 10 mM Tris @H 8.0), 1 mM EDTA.

For Southern blot analysis, the genomic DNA was digested with either BamHI or &al, subjected to electrophoresis, transferred to a nylon membrane, and hybridized to a random-primed radio- labeled probe corresponding to either the 3 ‘-end of SAG1 or a fragment of pBluescript, respectively. The hybridized membrane was examined by both autoradiography and phosphoimaging (Mo- lecular Dynamics).

PCR amplification of the genomic DNA was performed using two oligonucleotides (oligos) syn- thesized by Macromolecular Resources. The oligos correspond to sequences found within the ORF of SAG1 (sense: 5 ‘GCATCAAGGGAG- ACGACG) and in the 3’-UTR (anti-sense: 5’- CAAGCCACAGCGGAACAA) and are mapped in Fig. 1. The amplification reactions were performed in a 50 ~1 reaction volume with Perkin Elmer 10x buffer, 2.5 mM MgC12, 100 mM deoxynucleoside triphosphates (Promega), 10 pM of each oligo, and 1 pg of genomic DNA overlaid with paraffin oil (J.T. Baker Chemical Co.). The DNA was amplified for 25 cycles in a Perkin Elmer thermal cycler 480. After an initial 5-min incuba- tion at 94°C each cycle consisted of 1 min of denaturation at 94”C, l-2 min at the annealing temperature of 55”C, and a I-min extension at 72°C. The final extension step continued an additional 10 min.

3. Results and discussion

The influence of REM1 on transformation frequencies in the RH-strain of Toxoplasma was investigated using 100 units of restriction enzyme added to 20 pg of a plasmid encoding the select-


able marker chloramphenicol acetyltransferase (CAT, see pT/230 in Fig. 1) immediately prior to electroporation. Due to the slow nature of chloramphenicol selection [5], Toxoplasma required at least three passages in the drug to eliminate parasites not stably expressing CAT. This prevented us from obtaining accurate transformation frequen- ties and limited the results to comparisons between different systems. Initial experiments were performed using BumHI-linearized constructs with and without the addition of BarnHI enzyme. Following chloramphenicol selection, we repeatedly observed a 2-5-fold elevation in the number of REM1 transformants when compared to the transfection without added enzyme. Since the frequency of BumHI sites may present pro-

blems when working with genomic libraries, we also examined the influence of the rare cutting restriction enzyme NotI. Although based upon the ligation-repair model [1,7] one would expect a lower frequency due to the rarity of Nor1 sites within the genome, this enzyme enhanced the effl- ciency of transformation of No&linearized plasmid by 430-fold (Fig. 2). The difference in efficiency between Not1 and BarnHI REM1 has been reproduced four times with Not1 generating between 29 and 46 times more transformants than BarnHI. The enhancement by both BarnHI- and AN-REM1 is dependent on enzymatic activity as no significant increase in transformation frequency was observed using heat-inactivated restriction enzymes (data not shown). As the enzyme did not

Endogenous SAG1

(1) pSAG-tm

pSAG-set

pSAG+gal

pT1230

pT1230*

f______________+ 1 SAG1 m ______________ + BarnHI + t

Non

&SI SAG1 Southern probe

t PCR primers

- -. Genomic DNA BamHl

Nod [7 ORF

Bqalactosidase

q GPI

I TM

= SAG1 flanking sequence

m TUB promoter

- pBluescript

Fig. 1. Constructs, probe, and PCR primers used in the REM1 analyses (with endogenous SAGI for comparison). The indicated region of the endogenous SAG1 was used as the probe for the Southern blot in Fig. 3. The PCR primers were used to verify the acquisition of the altered SAGI constructs in clones following co-transfection. The pT/230 and pT/230* constructs, containing CAT driven by a SOO-bp tubulin promoter region, were used in the experiments testing the influence of REM1 on the transformation frequencies of Toxoplasma. These constructs differ only in the presence (pT/230) or absence (pT/230*) of the unique Not1 site. The pT/230 construct was also used as the selectable marker for the co-transfection experiment. The other three constructs (pSAG-tm, pSAG-see and pSAG-flgal) served as non-selectable markers in the co-transfection experiment. All of the constructs (including the CAT constructs) possessed the same 310-bp SAG1 3 ‘-UTR and the three non-selectable markers contained the same SAG1 promoter region. The two SAG1 constructs differed from each other and the endogenous copy at the 3 ‘-end of the open reading frame (ORF). These differences allowed detection of the altered genes based on the migration of both DNA and protein. The constructs are shown linearized at an arbitrary site in the vector sequence. (1) designates an altered pBluescript backbone in pSAG-tm resulting in a distinct restriction map when digested with RFaI (Fig. 5). The alteration does not appear to affect either the expression of the inserted SAG1 sequences or the functionality of pBluescript (i.e., copy number or ampicillin resistance).

M. Black et al. /Molecular and Biochemical Parasitology 74 (1995) 55-63 59

Iwo

plaque forming

units 100

Fig. 2. Comparison of transformation frequencies with Notl,

BarnHI, or no enzyme added to electroporations. In three of the transfections, a derivative of pTl230 (pT/230*) was used in which the unique NorI site was destroyed (described in methods). The absence of this restriction site is indicated by a (-) sign. Results are shown for parasites diluted 16-fold before plaquing.

demonstrate a significant influence on the survival or growth rate of the parasites immediately following transfection (i.e., in the absence of selection), the enhancement appears to be mediated through an increase in the actual transformation event(s) and not merely in the recovery after electroporation.

Transformation of pT/230 linearized with BumHI showed a 2.7-fold greater efficiency vs. Not1 linearized DNA when the respective enzymes were not included in the electroporation (Fig. 2). The basis of this has not been further investigated, but may be due to differences in the stability of the linearized DNA because of the single-stranded overhanging DNA characteristic of the different enzymes (‘GGCC’ for Not1 and ‘GATC’ for BumHI).

Unexpectedly, we find that it is not necessary for the enzyme used for linearization of the plasmid to be the same as that used in the electroporation. This was demonstrated by destroying the Not1 site in the pT/230 construct, linearizing this DNA with BarnHI, and adding Nor1 enzyme to the electroporation. In contrast to the situation in Dic- tyostelium (where compatible DNA ends are

necessary; [7]), we found that adding Not1 to the BumHI-linearized DNA still resulted in over a 90- fold increase in transformation when compared to the transfection of BarnHI-linearized DNA alone (Fig. 2). Indeed, even the undigested form of this No&deficient construct showed an - lOO-fold enhancement in the presence of Not1 compared to the same construct without added enzyme (Fig. 2).

To determine if REM1 influences the integration of pT/230 in T. go&ii in a similar manner as described in Saccharomyces and Dictyostelium, we examined the frequency of insertions into genomic restriction sites corresponding to the enzyme added to the electroporation. Genomic DNA from 22 REMI-transformed clones derived from three separate experiments (each using linearized DNA with ends compatible to the enzyme added) was digested with the enzyme used in the electroporation and analyzed by Southern blot. Of these 22 clones, only three demonstrated bands that co- migrated with the linearized construct used in the transfection (data not shown). Again, this result conflicts with the ligation repair model where a majority of the transformants would be expected to possess competent restriction sites tlanking the integrated DNA. The mechanism by which REM1 exerts its effect in Toxoplasma has not been determined here, but the data are consistent with the in- duction of a more general recombinogenic mechanism. Interestingly, it has been reported that some restriction enzymes possess a number of similarities to enzymes that catalyze site-specific recombination [ 17,181. Although this has not been further investigated in this report, the differences seen between BumHI and Not1 may not be based solely on their ability to cut DNA, but some other attribute of the enzyme which has not yet been identified.

Using REM1 with NotI, we have found that the increased transformation frequency can be used to

co-transfect multiple plasmids, of which only one expresses a selectable marker. In this experiment, we co-transfected the RH-strain of Toxoplasma with four NotI-linearized plasmids (Fig, 1) each encoding one of the following: (1) the selectable CAT marker (pT/230) [19J, (2) a &galactosidase reporter (pSAG-Bgal) [ 141, (3) a version of the normally GPI-anchored surface antigen SAG1 that


possesses a C-terminal truncation (pSAG-set) (Soldati and Boothroyd, unpublished), and (4) a longer trans-membrane version of this same protein (pSAG-tm) (Seeber and Boothroyd, unpublished). To increase the probability of stable transformants expressing the non-selectable markers, we used a 1:20:20:20 mass ratio ( - 1:15:20:20 molar ratio) of the four plasmids, respectively.

From approximately 1.5 x lo4 stable chloramphenicol-resistant plaques obtained, ten clones (Tl-TlO) were arbitrarily chosen for further analysis. As expected, all ten of the clones expressed high levels of CAT activity (Table 1). To determine which of these clones had also acquired one or more of the other three plasmids, each was analyzed for specific attributes characteristic of the non-selective markers. The enzymatic activity

Table 1 Attributes of ten clones of Toxoplasma gondii co-transfected with pT/230, pSAG-set, pSAG-tm, and pSAG-Bgal using nod REM1

pTl230’ pSAG- pSAG-tm b pSAG-see b Bgala

Tl + T2 + T3 + T4 + TS + T6 + Tl + T8 + T9 + T10 +

+ +C + += -

-

+ + -

_d _d

+ - + + + + + + -I- +

+ + +

a+/- indicates the presence or absence of enzymatic activity resulting from the uptake of pT/230 and pSAG-Bgal as determined by assays described in methods. bExcept where noted, +/- indicates the presence or absence of DNA and/or protein corresponding to the presence of pSAG- tm and pSAG-set as determined by Southern, western and PCR analyses. CAn altered (likely truncated) form of the pSAG-tm as supported by Southern blot, western blot, and PCR analyses. din very long exposures, faint bands appeared in TS migrating similar to the pSAG-tm and pSAG-set bands. This is likely to be a result of the ‘clone’ being, in fact, a mixed population (with the vast majority having the described TS attributes) due either to a cloning failure or a loss of the sequences in a fraction of the clonal population.

of /3-galactosidase was detected in seven of the ten clones obtained. The presence of the two SAG1 constructs was assessed by Southern blot analysis of BarnHI-digested genomic DNA probed with a radio-labeled SAG1 fragment (see Fig. 1 for probe location). The SAG1 constructs were discrim- inated from each other and the endogenous copy by differences in the length of their respective 3 ‘- ends (see map in Fig. 1). The results (Fig. 3) demonstrate that at least six out of the ten clones possess pSAG-tm, and four of the ten have the pSAG-sec. The band migrating at approximately 3 kb in Tl and T2 may be the result of an alteration in one of the SAG1 constructs resulting in the loss of a BarnHI site.

Tl T2 T3 T.5 T7

Fig. 3. Identification of altered SAG1 constructs by Southern blot analysis. BamHI-digested genomic DNA of the ten chloramphenicol selected clones (Tl -TlO) was examined using a SAG1 probe (see map in Fig. I). The lanes labeled TM and Set were used as controls containing similarly digested genomic DNA from the B-mutant of strain PLK of T. gondii stably transformed with pSAG-tm and pSAG-set, respectively (note that the endogenous SAG1 band (-9 kb) produced upon BumHI digestion of DNA from this strain has a different mobility from the RH SAG1 allele (- 12 kb)). The RH lane was a control using a digest of genomic DNA from the untransformed parental RH-strain. The band migrating at - 3 kb in TI and T2 (SAGtma) is thought to be an altered form of pSAG-tm that has lost the downstream BarnHI site as a result of a recombinational event (discussed in text). The faint band migrating at -7 kb in T3 was seen in all the lanes (including parental RH control) in a longer exposure and is the result of non-specific hybridization.


The presence of pSAG-tm was confirmed in all six Southern-positive clones by Western blot analysis (Fig. 4). The weak, but detectable, SAGtm bands in T8 and T9 contrast with the higher expression from other clones possessing the pSAG-tm plasmid. Since the intensity of the endogenous SAG1 band did not vary to such an extreme between the clones (Fig. 4), we conclude that the expression of this construct was repressed. While the basis for this inhibition was not further investigated, it could be due to the genomic site into which the plasmid integrated and/or the presence of a second, adjacent plasmid which pre- vious work has shown might adversely affect expression (Rim and Boothroyd, unpublished results).

As anticipated from the Southern results, the western blot for clones Tl and T2 showed a version of SAG1 that possessed an aberrant mobility (Fig. 4). Since the rate of migration is indicative of

Fig. 4. Expression of the modified SAG1 constructs as detected by Western blot analysis. Total lysates of each clone was examined along with the untransformed parental (RH) and B- mutant controls stably expressing pSAG-tm and pSAG-set (TM and Set). Although the trans-membrane form of SAG1 (SAGtm) could be clearly identified, the secreted SAG1 (SAGsec) migrated too close to the endogenous form and was not identified in this blot. Since the B-mutant does not express detectable levels of SAGI, both of the altered forms can be easily identified. The altered migration of SAGtm in clones TI and T2 (SAGtnP) may be the result of a recombinational truncation at the 3’-end of the construct (discussed in text). The expression of pSAG-tm in clones T8 and T9 was verified on an over-exposure and demonstrated similar results when examined independently to insure against contamination from adjacent wells (data not shown).

a protein with a higher molecular weight than the truncated or endogenous forms, yet smaller than the transmembrane version, we believe it is an altered version of pSAG-tm that has lost a portion of its C-terminus and quite possibly the entire 3’- UTR. This conclusion explains the distinct migration of its DNA in the Southern blot (loss of the downstream BarnHI in pBluescript). Our hypothesis is supported through the use of the polymerase chain reaction (PCR) to amplify the 3 ‘-ends of the distinct SAG1 genes using primers within the ORF and 3 ‘-UTR (see Fig. 1 for primer loci). As expected, clones Tl and T2 did not amplify the 3 ‘- end of pSAG-tm while the corresponding region of the endogenous SAG1 was amplified normally (data not shown). This result would indicate that one or both of the sequences in the 3’-end in pSAG-tm complementary to the primers are either missing or altered so as to prevent proper amplification of the region. The PCR-amplification of the other eight clones correlated with the Southern blot analysis for endogenous, pSAG-see, and pSAG-tm versions of the 3’-end of SAG1 (data not shown).

Western blot analysis was also used to determine if the pSAG-set constructs were expressed within the clones positive for this plasmid by Southern blot analysis. As a result of the small difference in migration between the truncated SAGsec and the endogenous form of SAG1 (Fig. 4), SAGsec could not be resolved using total lysates. Although we were able to separate the two forms by performing a series of detergent extrac- tions, only T7 and TlO showed weak but detectable levels of SAGsec (data not shown). This result was not unanticipated since the positive control (a stable transformant expressing this gene) also demonstrated low levels of protein by this assay. While this limited detection may be the result of insufficient recovery of the protein due to the secretion of this normally GPI-anchored protein (Soldati and Boothroyd, unpublished results), the absence of SAGsec in T8 and T9 may also have been due to circumstances described above for the low expression of pSAG-tm in these same clones.

Taken together, the ten clones consist of two that possess pT/230 only, one that has also integrated pSAG-tm, three that also have both pSAG-


tm and pSAG+gal, and four that appear to have stably acquired all four of the constructs (Table 1). To determine if these latter four clones are pro- geny of a single transformed parasite, a Southern blot was performed using a &zI genomic digest with a pBluescript fragment as a probe. From this analysis, we find that all four strains appear to be independent clones as indicated by unique band patterns attributed to distinct sequences flanking one or more of the integrated constructs (Fig. 5). Using this and other diagnostic genomic digests, we also determined that co-integration of more than one DNA into a single locus had occurred in the eight clones obtaining one or more unselected markers (Tl, T2, T4 and T6-TlO). This was identified by the presence of bands corresponding to the expected sizes generated if head-to-head or

Fig. 5. Independence of four co-transfectants as detertnined by Southern blot analysis. RraI-digested genomic DNA of the co- transfected clones possessing all four unselected markers (T7-TlO) and the parental RH-strain (RH) was examined using a radio-labeled fragment of pBluescript as a probe. The distinct bands represent heterogeneity of the Rsal restriction sites adjacent to the integrated constmcts resulting in the production of fragments of varying lengths. Since none of the lanes are identical, we conclude that while there appear to be striking similarities (see text), the clones are not siblings but are the result of separate genomic integration events. The band corresponding to a 1. I-kb DNA is an internal RFaI fragment from the pBluescript backbone of pT/230, pSAG-set and pSAG-@gal and can be seen in each of the transformed clones (Tl-T6 not shown). The 1%kb band is an internal RsaI fragment from a rearranged pBluescript backbone in the original pSAG-tm (see legend in Fig. 1) and is found only in clones possessing this con- stmct. The proposed origins of the 3.0- and 2.2-kb bands are discussed in the text.

head-to-tail co-integrations occurred (e.g., 3.0 and 2.2 kb bands in Fig. 5). The co-integration of two or more plasmids may have influenced the distinct expression patterns of pSAG-tm and pSAG-set in clones T8 and T9 by either direct interference or indirect inhibition resulting from adjacent genomic activity as described above. The copy number of the unselected SAG1 constructs appeared to range from one to three per genome based on phospho-imaging analysis of a Southern blot when compared to the intensity of the endogenous copy (data not shown).

A Southern blot of No&digested genomic DNA from the ten clones showed only one clone (T3) with a construct-sized band using pBluescript as a probe (data not shown). This result is consistent with the data above suggesting that the constructs either are not integrating into genomic Not1 sites, or the sites are destroyed during the process. As eight out of the ten clones demonstrated co- integration events, one might expect that a Not1 digest would excise at least one construct if three or more co-integrated into a single locus. Since this was not demonstrated, we conclude that either the Not1 sites are destroyed upon co-integration or, less likely, that only two constructs co-integrated at any single site in each of the eight clones.

In conclusion, while restriction enzyme- mediated integration can substantially elevate the frequency of transformation in T. gondii, it does not appear to function in the same manner as described in organisms such as Saccharomyces and Dictyostelium. While we did not investigate the mode of integration, we suggest that REM1 may elicit the activation of recombinogenic DNA repair machinery resulting in the elevation of illegitimate recombination events. In addition to elevating the transformation frequency, REM1 also appears to facilitate the detection of co- integration events; resulting in the ability to trans- form several non-selectable markers by co- transfection under a single form of selection. This procedure may prove useful in increasing the rate of transformation in other recently developed systems such as Plasmodium [20,21] and Entameoba [22,23] and in genetically engineering strains to receive multiple genes when only a limited number of selectable markers are available.


Acknowkdgements 191

We thank Drs. Ian Manger, Keith Wilson, Adrian Hehi, Jeff Cirilio, and other members of our lab for helpful comments and Jeff van Wye for provision of stains. This work was supported in part by grants to John Boothroyd from the NIH (AI21423 and A130230) with fellowship support from the Howard Hughes Medical Institute (Michael Black), the AIDS-Stipendienprogramm, Deutsches Krebsforschungszentrm (Frank Seeber), the European Molecular Biology Organi- zation and the Swiss National Science Foundation (Dominique Soldati), and the NIH and University of California Universitywide AIDS Research Pro- gram (Kami Kim).

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Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii