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• W1994016728 abstract "To dissect the contributions of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and adenosine kinase (AK) to purine salvage in Leishmania donovani, null mutants genetically deficient in HGPRT and/or APRT were generated by targeted gene replacement in wild type cells and preexisting mutant strains lacking either APRT or AK activity. These knockouts were obtained either by double targeted gene replacement or by single gene replacement followed by negative selection for loss-of-heterozygosity. Genotypes were confirmed by Southern blotting and the resultant phenotypes evaluated by enzymatic assay, resistance to cytotoxic drugs, ability to incorporate radiolabeled purine bases, and growth on various purine sources. All mutant strains could propagate in defined growth medium containing any single purine source and could metabolize exogenous [3H]hypoxanthine to the nucleotide level. The surprising ability of mutant L. donovani lacking HGPRT, APRT, and/or AK to incorporate and grow in hypoxanthine could be attributed to the ability of the parasite xanthine phosphoribosyltransferase enzyme to salvage hypoxanthine. These genetic studies indicate that HGPRT, APRT, and AK, individually or in any combination, are not essential for the survival and growth of the promastigote stage of L. donovani and intimate an important, if not crucial, role for xanthine phosphoribosyltransferase in purine salvage. To dissect the contributions of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and adenosine kinase (AK) to purine salvage in Leishmania donovani, null mutants genetically deficient in HGPRT and/or APRT were generated by targeted gene replacement in wild type cells and preexisting mutant strains lacking either APRT or AK activity. These knockouts were obtained either by double targeted gene replacement or by single gene replacement followed by negative selection for loss-of-heterozygosity. Genotypes were confirmed by Southern blotting and the resultant phenotypes evaluated by enzymatic assay, resistance to cytotoxic drugs, ability to incorporate radiolabeled purine bases, and growth on various purine sources. All mutant strains could propagate in defined growth medium containing any single purine source and could metabolize exogenous [3H]hypoxanthine to the nucleotide level. The surprising ability of mutant L. donovani lacking HGPRT, APRT, and/or AK to incorporate and grow in hypoxanthine could be attributed to the ability of the parasite xanthine phosphoribosyltransferase enzyme to salvage hypoxanthine. These genetic studies indicate that HGPRT, APRT, and AK, individually or in any combination, are not essential for the survival and growth of the promastigote stage of L. donovani and intimate an important, if not crucial, role for xanthine phosphoribosyltransferase in purine salvage. Protozoan parasites cause a variety of devastating and often fatal diseases in humans and their domestic animals. The treatment and control of parasitic diseases, however, is severely compromised by the dearth of effective and selective antiparasitic therapies. Many of the current antiparasitic drugs cause severe toxicity in the host, a predicament that can be attributed to lack of target specificity. Moreover, these drugs are potentially mutagenic and/or carcinogenic, they often require protracted courses with multiple drug administrations, and therapeutic unresponsiveness and drug resistance have exacerbated the necessity for new and improved antiparasitic agents. The institution of a rational therapeutic regimen for the treatment and prevention of parasitic diseases hinges upon exploitation of fundamental biochemical disparities between parasite and host. Perhaps the most striking metabolic discrepancy between parasites and humans is the purine pathway. Whereas mammalian cells can synthesize the purine heterocycle de novo, all protozoan parasites studied thus far are auxotrophic for purines (1Berens R.L. Krug E.C. Marr J.J. Marr J.J. Muller M. Biochemistry of Parasitic Organisms and Its Molecular Foundations. Academic Press Limited, London1995: 89-117Google Scholar). As a consequence, each genus of parasite has evolved a unique complement of purine salvage enzymes that enable the organism to scavenge host purines. Unique features of the purine salvage pathway of Leishmania andTrypanosoma constitute the basis for the susceptibility of these genera to several pyrazolopyrimidine analogs of naturally occurring purine bases and nucleosides (2Marr J.J. Berens R.L. Mol. Biochem. Parasitol. 1983; 7: 339-356Crossref PubMed Scopus (90) Google Scholar, 3Ullman B. Pharmaceut. Res. 1984; 1: 194-203Crossref PubMed Scopus (25) Google Scholar). The intact parasites efficiently metabolize these analogs to the nucleotide level, whereas mammalian cells are essentially incapable of these metabolic transformations. One of these pyrazolopyrimidines, allopurinol (4-hydroxypyrazolo[3,4]pyrimidine, HPP), 1The abbreviations used are: HPP, allopurinol; AK, adenosine kinase; APP, 4-aminopyrazolopyrimidine; APRT, adenine phosphoribosyltransferase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; HGXPRT, hypoxanthine-guanine-xanthine phosphoribosyltransferase; kb, kilobase pair(s); LOH, loss-of-heterozygosity; PCR, polymerase chain reaction; PRT, phosphoribosyltransferase; XPRT, xanthine phosphoribosyltransferase.1The abbreviations used are: HPP, allopurinol; AK, adenosine kinase; APP, 4-aminopyrazolopyrimidine; APRT, adenine phosphoribosyltransferase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; HGXPRT, hypoxanthine-guanine-xanthine phosphoribosyltransferase; kb, kilobase pair(s); LOH, loss-of-heterozygosity; PCR, polymerase chain reaction; PRT, phosphoribosyltransferase; XPRT, xanthine phosphoribosyltransferase.a drug that is nontoxic to humans and is widely used in the treatment of hyperuricemia and gout (4Palella T.D. Fox I.H. Scriver C.S. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Diseases. 6th Ed. McGraw Hill Book Co., New York1989: 965-1006Google Scholar), has demonstrated significant therapeutic efficacy in patients with either cutaneous leishmaniasis (5Martinez S. Marr J.J. N. Engl. J. Med. 1992; 326: 741-744Crossref PubMed Scopus (151) Google Scholar) or chronic Chagas disease (6Gallerano R.H. Sosa R.R. Marr J.J. Am J. Trop. Med. Hyg. 1990; 43: 159-166Crossref PubMed Scopus (73) Google Scholar). Leishmania donovani, the causative agent of visceral leishmaniasis, is a digenetic parasite that exists as an extracellular promastigote within the insect vector, members of the phlebotomine sandfly family, and as a nonmotile intracellular amastigote within the phagolysosome of macrophages and other cells of the reticuloendothelial system of the mammalian host. L. donovani expresses a number of enzymes capable of converting preformed purines directly to nucleotides. These enzymes include hypoxanthine-guanine phosphoribosyltransferase (HGPRT; IMP:pyrophosphate phosphoribosyltransferase; EC 2.4.2.8), adenine phosphoribosyltransferase (APRT; AMP:pyrophosphate phosphoribosyltransferase; EC 2.4.2.7), xanthine phosphoribosyltransferase (XPRT; XMP:pyrophosphate phosphoribosyltransferase; EC 2.4.2.22), and adenosine kinase (AK).Leishmania also contain a plethora of purine interconversion enzymes including nucleosidases, phosphorylases, deaminases, and IMP branchpoint enzymes (1Berens R.L. Krug E.C. Marr J.J. Marr J.J. Muller M. Biochemistry of Parasitic Organisms and Its Molecular Foundations. Academic Press Limited, London1995: 89-117Google Scholar). Overall, the purine pathway is divagating and intricate, and this metabolic complexity and the apparent overall diploid nature of the parasite (7Cruz A. Beverley S.M. Nature. 1990; 348: 171-173Crossref PubMed Scopus (193) Google Scholar, 8Iovannisci D.M. Goebel D. Kaur K. Allen K. Ullman B. J. Biol. Chem. 1984; 259: 14617-14623Abstract Full Text PDF PubMed Google Scholar, 9Iovannisci D.M. Beverley S.M. Mol. Biochem. Parasitol. 1989; 34: 177-188Crossref PubMed Scopus (83) Google Scholar) has hindered a thorough characterization of the purine pathway in the parasite by straightforward biochemical and genetic approaches. The ability of Leishmania to undergo a high rate of homologous gene replacement (7Cruz A. Beverley S.M. Nature. 1990; 348: 171-173Crossref PubMed Scopus (193) Google Scholar, 10Tobin J.F. Laban A. Wirth D.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 864-868Crossref PubMed Scopus (34) Google Scholar, 11Cruz A. Coburn C.M. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7170-7174Crossref PubMed Scopus (279) Google Scholar, 12Curotto de Lafaille M.A. Wirth D.F. J. Biol. Chem. 1992; 267: 23839-23846Abstract Full Text PDF PubMed Google Scholar) and to take up foreign DNAs after transfection (13Laban A. Tobin J.F. de Lafaille M.A.C. Wirth D.F. Nature. 1990; 343: 572-574Crossref PubMed Scopus (125) Google Scholar, 14Kapler G.M. Coburn C.M. Beverley S.M. Mol. Cell. Biol. 1990; 10: 1084-1094Crossref PubMed Scopus (357) Google Scholar) now permits an assessment of specific gene function by targeted gene replacement and facilitates the genetic dissection of complex metabolic pathways such as that for purine acquisition. As both the HGPRT (15Allen T.E. Hwang H.-Y. Jardim A. Olafson R. Ullman B. Mol. Biochem. Parasitol. 1995; 73: 133-145Crossref PubMed Scopus (37) Google Scholar) and APRT (16Allen T.E. Hwang H.-Y. Jardim A. Ullman B. Mol. Biochem. Parasitol. 1995; 74: 99-103Crossref PubMed Scopus (24) Google Scholar) genes and their flanking regions have been isolated, we have examined the relative contributions of HGPRT, APRT, and AK to purine salvage inL. donovani promastigotes by sequentially eliminating eachHGPRT and APRT allele from wild type cells and from preexisting mutants lacking either APRT (8Iovannisci D.M. Goebel D. Kaur K. Allen K. Ullman B. J. Biol. Chem. 1984; 259: 14617-14623Abstract Full Text PDF PubMed Google Scholar) or AK (17Iovannisci D.M. Ullman B. Mol. Biochem. Parasitol. 1984; 12: 139-151Crossref PubMed Scopus (39) Google Scholar) activity by homologous gene replacement and/or direct selection. These strains were created either by replacing each allele sequentially with independent drug resistance markers or by disrupting the first allele with one drug resistance marker and selecting for loss-of-heterozygosity (LOH) with drug pressure to obtain the homozygous null mutant. The new alleles in the heterozygotes and homozygotes were examined by Southern blotting, and the resultant phenotypes of the mutant strains have been evaluated for enzymatic activities, ability to take up purines, drug resistance profiles, and growth properties. [14C]Hypoxanthine, [14C]guanine, [14C]adenine, and [14C]xanthine, all at 56 mCi/mmol, and [3H]adenosine at 32.5 Ci/mmol were obtained from Moravek Biochemicals (Brea, CA). [α-32P]dCTP (3000 Ci/mmol) and [α-35S]dATP (1320 Ci/mmol) were bought from NEN Life Science Products. All restriction and DNA modifying enzymes were acquired from either New England Biolabs, Inc. or Life Technologies, Inc., and Thermus flavus DNA polymerase was purchased from Epicentre Technologies (Madison, WI). The pX63-NEO and pX63-HYG drug resistance cassettes were furnished by Dr. Stephen Beverley (Harvard Medical School). The sources of all other chemicals and reagents were of the highest quality commercially available. Promastigotes of the Sudanese 1S strain ofL. donovani were grown in DME-L culture medium (18Iovannisci D.M. Ullman B. J. Parasitol. 1983; 69: 633-636Crossref PubMed Scopus (123) Google Scholar). Unless otherwise specified, DME-L contains 100 μm xanthine as a purine source. Clonal lines of L. donovani were isolated as colonies on semi-solid DME-L medium containing appropriate selective agent as indicated (18Iovannisci D.M. Ullman B. J. Parasitol. 1983; 69: 633-636Crossref PubMed Scopus (123) Google Scholar). Initial recipient strains for the transfection experiments reported here included the wild type DI700 line and its two clonal derivatives, TUBA2 and APPB2A3. The isolation and biochemical characterizations of the AK-deficient (ak−) TUBA2 (17Iovannisci D.M. Ullman B. Mol. Biochem. Parasitol. 1984; 12: 139-151Crossref PubMed Scopus (39) Google Scholar) and the APRT-deficient (aprt−) APPB2A3 (8Iovannisci D.M. Goebel D. Kaur K. Allen K. Ullman B. J. Biol. Chem. 1984; 259: 14617-14623Abstract Full Text PDF PubMed Google Scholar) strains have been reported previously. TUBA2 and APPA2B3 cells and all of their progeny were continuously maintained in either 1 μm tubercidin, a cytotoxic AK substrate (17Iovannisci D.M. Ullman B. Mol. Biochem. Parasitol. 1984; 12: 139-151Crossref PubMed Scopus (39) Google Scholar), or 100 μm4-aminopyrazolopyrimidine, a subversive substrate of APRT (8Iovannisci D.M. Goebel D. Kaur K. Allen K. Ullman B. J. Biol. Chem. 1984; 259: 14617-14623Abstract Full Text PDF PubMed Google Scholar), respectively, to ensure retention of the selected phenotype. For the genetic manipulations of the HGPRT and APRT loci described here, DI700, TUBA2, and APPB2A3 cell lines are designated DI700:H+/+A+/+, TUBA2:H+/+A+/+, and APPB2A3:H+/+, respectively, where H refers to HGPRT, A to APRT, and + is the wild type allele (see Table I). No allelic nomenclature is given for the APRT locus of APPB2A3 cells, as the genetic basis for the APRT deficiency in this strain has not been characterized. Sensitivities of wild type and strains created by targeted gene replacement to HPP, 4-aminopyrazolo[3,4]pyrimidine (APP), 4-thiopurinol, and HPP riboside were performed as described (8Iovannisci D.M. Goebel D. Kaur K. Allen K. Ullman B. J. Biol. Chem. 1984; 259: 14617-14623Abstract Full Text PDF PubMed Google Scholar,17Iovannisci D.M. Ullman B. Mol. Biochem. Parasitol. 1984; 12: 139-151Crossref PubMed Scopus (39) Google Scholar). Protocols utilized by this laboratory for obtaining parasite DNA, for Southern blotting, and for nucleotide sequencing have all been described previously (19Wilson K. Collart F. Huberman E. Stringer J. Ullman B. J. Biol. Chem. 1991; 266: 1665-1671Abstract Full Text PDF PubMed Google Scholar, 20Hanson S. Adelman J. Ullman B. J. Biol. Chem. 1992; 267: 2350-2359Abstract Full Text PDF PubMed Google Scholar). The L. donovani HGPRT (15Allen T.E. Hwang H.-Y. Jardim A. Olafson R. Ullman B. Mol. Biochem. Parasitol. 1995; 73: 133-145Crossref PubMed Scopus (37) Google Scholar) and APRT (16Allen T.E. Hwang H.-Y. Jardim A. Ullman B. Mol. Biochem. Parasitol. 1995; 74: 99-103Crossref PubMed Scopus (24) Google Scholar) were isolated from cosmids as 3.5-kb EcoRI and 5.4-kbBamHI-XbaI restriction fragments, respectively. Flanking regions of the two genes were identified by restriction mapping and Southern blotting. An additional 5.2-kbSalI-EcoRI (21Hwang H.-Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) DNA located immediately 5′ to the 3.5-kb EcoRI DNA was also obtained from theHGPRT-containing cosmid to amplify the 5′-flanking region ofHGPRT for construction of gene targeting vectors. The flanking regions from the HGPRT and APRT loci and the oligonucleotides used for their amplification by the polymerase chain reaction (PCR) have been reported previously (21Hwang H.-Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The construction and authentication of the pX63-NEO-Δhgprt and pX63-HYG-Δaprt plasmids employed in the allelic replacements of the HGPRT and APRT loci, respectively, have also been described (21Hwang H.-Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). pX63-NEO-Δhgprt contains a 1.7-kb 5′ and a 1.8-kb 3′-flanking region from the L. donovani HGPRTencompassing the neomycin phosphotransferase (NEO) gene, while pX-HYG-Δaprt includes a 1.4-kb 5′ and a 1.5-kb 3′ flank of the L. donovani APRT circumscribing the hygromycin phosphotransferase (HYG) marker. To generate aHYG construct for replacing the HGPRT locus, the 1.7-kb 5′ and 1.8-kb 3′ flanks employed for making pX63-NEO-Δhgprt were reamplified using the PCR conditions described by Hwang et al. (21Hwang H.-Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and ligated into the appropriate restriction sites within pX63-HYG. The pX63-HYG-Δhgprt construct was verified for identity and orientation by limited restriction mapping and nucleotide sequencing. Parasites were transfected using electroporation conditions reported previously (11Cruz A. Coburn C.M. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7170-7174Crossref PubMed Scopus (279) Google Scholar, 21Hwang H.-Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). All targeting constructs were cleaved from their plasmids by digestion withHindIII and BglII just prior to electroporation, isolated on agarose gels, and purified using the GeneClean Kit (Intermountain Scientific Corp., Bountiful, UT). Designation of the targeting DNAs originated from the plasmid nomenclature without the initial letter, e.g. X63-HYG-Δhgprtfrom pX63-HYG-Δhgprt. All transfected cells were grown for 24 h under nonselective conditions prior to the initiation of drug pressure. To generate lines heterozygous at either the HGPRT (hgprt/HGPRT) or APRT(aprt/APRT) locus, L. donovani promastigotes were transfected with the appropriate targeting construct, and heterozygotes were selected by the addition of either 50 μg/ml Geneticin (G418) or hygromycin B to liquid medium or 16 μg/ml G418 or 50 μg/ml hygromycin B to semi-solid medium. Clonal populations were selected on agar plates as described (18Iovannisci D.M. Ullman B. J. Parasitol. 1983; 69: 633-636Crossref PubMed Scopus (123) Google Scholar). The genotypes of the heterozygotes were determined by Southern blotting using appropriate protein coding and flanking probes. To generate homozygous null mutants (Δhgprt or Δaprt) from the heterozygotes, two strategies were employed. The first required a second round of transfection with an independent drug resistance cassette, i.e. double targeted gene replacement. Using this approach, both G418 and hygromycin were added to the plates to select for parasites in which the wild type allele in the heterozygous recipient had been specifically disrupted by homologous recombination of the second targeting construct. The second protocol for selecting homozygous null mutants at either theHGPRT or APRT loci was to select for LOH by plating in semi-solid agarose to which a toxic substrate of the encoded gene product had been added. Specific selective conditions were 1–3 mm HPP to obtain Δhgprt clones and 100 μm APP to obtain Δaprt clones. Selections for LOH at the HGPRT and APRT loci included G418 and/or hygromycin, as specified, in the medium to retain drug resistance cassettes that had been previously integrated at the appropriate locus/loci by targeted gene replacement. All L. donovani transformants that had integrated copies of either X63-NEO-Δhgprt, X63-HYG-Δhgprt, or X63-HYG-Δaprt into the relevant loci were maintained continuously under selective pressure in the drugs for which they contained resistance markers. In addition, knockout cell lines created by single targeted gene replacement as described above were also grown perpetually in the agent in which they were selected,i.e. 2 mm HPP or 100 μm APP as appropriate. Exponentially growing L. donovaniwere harvested in 50 mm Tris, pH 8.0, containing 5 mm MgCl2, and 1 mm dithiothreitol and lysed either by sonication or by three rounds of freeze-thawing. HGPRT (22Allen T. Henschel E.V. Coons T. Cross L. Conley J. Ullman B. Mol. Biochem. Parasitol. 1989; 33: 273-281Crossref PubMed Scopus (29) Google Scholar), APRT (22Allen T. Henschel E.V. Coons T. Cross L. Conley J. Ullman B. Mol. Biochem. Parasitol. 1989; 33: 273-281Crossref PubMed Scopus (29) Google Scholar), and AK (17Iovannisci D.M. Ullman B. Mol. Biochem. Parasitol. 1984; 12: 139-151Crossref PubMed Scopus (39) Google Scholar) activities were measured in 20,000 × g supernatants as reported. The rate of [14C]hypoxanthine incorporation into phosphorylated metabolites in the absence or presence of excess nonradiolabeled purines was ascertained at room temperature using a slight modification of the DE-81 filter disk method outlined by Iovannisci et al. (17Iovannisci D.M. Ullman B. Mol. Biochem. Parasitol. 1984; 12: 139-151Crossref PubMed Scopus (39) Google Scholar). 108 parasites were harvested by centrifugation, washed with phosphate-buffered saline, and resuspended in 1.0 ml of modified DME-L growth medium lacking the hemin and xanthine components prior to the addition of radiolabel and purine additives. [14C]Hypoxanthine and nonradiolabeled purines were present at 1.8 μm and 100 μm, respectively, in the uptake assays. The assay was terminated by removing 100-μl aliquots of (107) cells, diluting them in ice cold phosphate-buffered saline, and subjecting them to centrifugation. The pelleted cells were washed twice in 1.0 ml of phosphate-buffered saline, lysed in 1% Triton X-100, and blotted onto a 1.5-cm2 piece of DE-81 impregnated paper. The disks were washed as described (17Iovannisci D.M. Ullman B. Mol. Biochem. Parasitol. 1984; 12: 139-151Crossref PubMed Scopus (39) Google Scholar). A flow chart describing the derivation and lineages of all of the mutant strains that were created as a result of targeted gene replacement and/or direct selection in cytotoxic substrate is depicted in Fig.1. Three parental recipient clonal cell lines were employed for the transfection experiments, DI700 wild type cells, TUBA2 ak− cells, and APPB2A3 aprt− cells. The three parental cell lines are designated DI700:H+/+A+/+, TUBA2:H+/+A+/+, and APPB2A3:H+/ n, respectively (Fig. 1), as theirHGPRT and APRT loci have not been targeted by gene replacement vectors. Heterozygotes (hgprt/HGPRT) in which a single wild type HGPRT allele was disrupted by targeted gene replacement approaches were obtained from DI700 (DI700:H+/ n A+/+) and TUBA2 (TUBA2:H+/ n A+/+) cells with X63-NEO-Δhgprt as the targeting vector and from APPA2A3 (APPB2A3:H+/ h) cells with X63-HYG-Δhgprt. Homozygous null mutants (Δhgprt) were then obtained from the threehgprt/HGPRT lines by two independent strategies, either by targeting with the appropriate construct containing the second drug resistance marker (DI700:H n/h A+/+, TUBA2:H n/h A+/+, and APPB2A3:H h/n) or by direct negative selection (DI700:H n/n A+/+ and TUBA2:H n/n A+/+) for LOH (see Fig. 1). Nomenclature for all the Δhgprt cell lines shown in Fig. 1 conforms to that employed for the heterozygotes. The ability to generate Δhgprt null mutants after only a single round of transfection permitted the creation of L. donovani cell lines in which both wild type alleles of theHGPRT and APRT loci had been displaced, each with a single targeting construct. Cell lines heterozygous at the APRT locus (aprt/APRT) were thus created from the DI700:H n/n A+/+ and TUBA2:H n/n A+/+ cell lines, as well as from the parental TUBA2:H+/+A+/+strain by targeted gene replacement using the X63-HYG-Δaprt construct. These derivatives were designated DI700:H n/n A+/ h, TUBA2:H n/n A+/ h, and TUBA2: H+/+A+/ h, respectively (Fig.1). Δaprt clonal progeny, DI700:H n/n A h/h, TUBA2:H n/n A h/h, and TUBA2:H+/+A h/h, were then selected for LOH from the three aprt/APRT heterozygotes in 100 μm APP (Fig. 1). Southern blot analysis verified the existence of the new alleles that had been created after either double or single targeted gene replacement. The maps of the genomic loci forHGPRT and APRT and the novel alleles created by homologous recombination of X63-NEO-Δhgprt into theHGPRT locus and X63-HYG-Δaprt into theAPRT locus have been reported previously (21Hwang H.-Y. Gilberts T. Jardim A. Shih S. Ullman B. J. Biol. Chem. 1996; 271: 30840-30846Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The X63-HYG-Δhgprt construct and its rearranged allele after insertion into the chromosome are displayed in Fig. 2. The maps of the HGPRT and APRT loci are also included in Fig. 2 to show the location of the probes employed in the Southern blotting experiments. The expected size differences of the wild type and disrupted HGPRT alleles in strains that were generated or employed for these studies can be visualized in Fig.3. The interrupted HGPRT alleles could be distinguished from the wild type allele by an altered EcoRI restriction pattern (Fig. 3). After cleavage of genomic DNA withEcoRI and hybridization to probe B derived from the 3′-flanking region of HGPRT (see Fig. 2), only the anticipated 4.6-kb EcoRI fragment from the wild type allele was discerned in the three parental, i.e, DI700:H+/+A+/+, APPB2A3:H+/+, and TUBA2:H+/+A+/+, strains in which theHGPRT locus was intact (see Fig. 3, probe B). Although the HGPRT was originally isolated as a 3.5-kbEcoRI fragment (see “Experimental Procedures”), the 4.6-kb EcoRI band is the expected size of the wild typeHGPRT allele, as the 3′ EcoRI restriction site of the cloned 3.5-kb fragment originates from the cosmid. In contrast, bands of 6.4 and 6.2 kb were observed in those strains in which anHGPRT allele had been targeted by either X63-NEO-Δhgprt or X63-HYG-Δhgprt, respectively (Fig. 3). Cell lines heterozygous for HGPRT (H+/ h or H+/ h) retained the 4.6-kb EcoRI fragment derived from the wild type locus, while HGPRT null mutants (H n/h or H n/n) lacked the wild type 4.6-kbEcoRI restriction fragment and exhibited only the expected 6.4- and 6.2-kb displacements by X63-NEO-Δhgprt and X63-HYG-Δhgprt, respectively (Fig. 3, probe B). The interruption of the wild type HGPRT allele was verified by hybridization of genomic DNA to probe Acorresponding to the coding region of HGPRT (see Fig. 2). The 4.6-kb EcoRI fragment was detected only in cell lines that were wild type or heterozygous at the HGPRT locus,i.e. the H+/+, H+/ n, and H+/ h strains and not in Δhgprt null mutants that were derived by either double targeted (H n/h) or single targeted (H n/n) gene replacement (Fig. 3, probe A).Figure 3Southern blot analysis of the HGPRTlocus in wild type and mutant strains. Genomic DNA samples from various strains of L. donovani created in these studies (see Fig. 1) were digested with EcoRI and hybridized toprobe A and probe B from the HGPRTlocus. The cells from which the genomic DNA samples were obtained are displayed above each lane in the electrophoretogram. Panel Ashows the Southern blots of DI700 and APPB2 cells and their progeny, whereas panel B displays the blots of TUBA2 and its derivatives. The locations of probes A and B are indicated in Fig. 2. The sizes of the restriction fragments hybridizing to the HGPRT and APRT probes are shown on theleft in base pairs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It should be noted that after targeting wild type cells with X63-NEO-Δhgprt or X63-HYG-Δhgprt, 23 of 24 clones isolated and analyzed by Southern blotting displayed only simple gene replacements, whereas 12 of 15 colonies isolated fromhgprt/HGPRT heterozygotes targeted with X63-HYG-Δhgprt exhibited complex genetic events other than simple gene replacements. These enigmatic genetic alterations in the anomalous cell lines were not analyzed in detail, and the reasons for the large discrepancies in the frequency by which various genetic events were observed during each round of transfection is unknown. However, genetic events that do not involve simple allelic replacements after homologous recombination of an extrachromosomal fragment have been observed previously in Leishmania spp. (7Cruz A. Beverley S.M. Nature. 1990; 348: 171-173Crossref PubMed Scopus (193) Google Scholar, 12Curotto de Lafaille M.A. Wirth D.F. J. Biol. Chem. 1992; 267: 23839-23846Abstract Full Text PDF PubMed Google Scholar, 23Cruz A.K. Titus R. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1599-1603Crossref PubMed Scopus (170) Google Scholar). Disruption of the wild type APRT locus in lines that were targeted by X63-HYG-Δaprt was also validated by Southern blot analysis (Fig. 4). aprt/APRTheterozygotes and Δaprt homozygous knockouts were only derived from the DI700 and TUBA2 lines (see Fig. 1), as APPB2A3 cells already lacked APRT activity (8Iovannisci D.M. Goebel D. Kaur K. Allen K. Ullman B. J. Biol. Chem. 1984; 259: 14617-14623Abstract Full Text PDF PubMed Google Scholar). Digestion of genomic DNA withSalI and BamHI and hybridization to probe D derived from the 5′-flanking region of APRT (Fig. 2) revealed the presence of the 3.5-kb SalI/BamHI restriction fragment from the wild type allele in all A+/+and A+/ h cells (Fig. 4, probe D). Theaprt/APRT heterozygotes contained an additional 1.4-kbSalI band (see Fig. 2) derived from the new allele created by integration of X63-HYG-Δaprt into the APRT locus. Hybridization of DNA from all of the presumptive Δaprtnull mutants to probe D revealed only the 1.4-kb band expected from the integration of the drug resistance cassette into both alleles of the APRT locus (Fig. 4, probe D). Genetic disruption of wild type APRT alleles was confirmed by hybridization of the same blot to probe C encompassing most of the APRT coding region (Fig. 4, probe C).APRT coding sequences existed only in cells containing one or two wild type APRT copies, whereas the knockout lines TUBA2:H+/+A h/h and TU" @default.
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