Stable amplification of a linear extrachromosomal DNA in mycophenolic acid-resistant Leishmania donovani

Molecular and Biochemical Parasitology, 55 (1992) 197-206

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© 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 01824

Stable amplification of a linear extrachromosomal D N A in mycophenolic acid-resistant Leishmania donovani Keith Wilson a, Stephen M. Beverley b and B u d d y U l l m a n a aDepartment of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR, USA; and bDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA (Received 12 May 1992; accepted 15 July 1992)

Pulsed field gel electrophoretic analysis of chromosomes of MPA 100 cells, a strain of Leishmania donovani that possesses an approx. 15-fold amplified IMP dehydrogenase (IMPDH) gene copy number, revealed a new 280-kb extrachromosomal DNA, IMPDH-280, that was not present in wild type parental cells. Southern blots of these pulsed field gels revealed that the vast majority of the amplified impdh genes were localized on IMPDH-280. In addition to the 700-kb wild type chromosome, the impdh probe also recognized a 740-kb chromosome in the MPA100 genome. The pulse time-dependent relative mobility of IMPDH-280 in pulsed field gels, the failure of limited 7-irradiation to generate a new discrete DNA fragment, and the susceptibility of IMPDH-280 to 2-exonuclease digestion, demonstrated that IMPDH-280 was a linear molecule. IMPDH-280 was also recognized by a telomere probe but not by fragments derived from amplified DNAs found in other drug-resistant Leishmania. IMPDH-280 and the drug resistance phenotype remained stable when MPA100 cells were propagated in the absence of drug for 2 years. The appearance of 1MPDH-280 in MPAI00 cells represents one of the first examples of an amplification of a linear extrachromosomal DNA element mediating drug resistance in Leishmania and the first instance of a linear DNA amplification that is stable in the absence of selective pressure. Key words: Le&hmania donovani; Gene amplification; Drug resistance; Extrachromosomal element; Pulsed field gel electrophoresis; IMP dehydrogenase

Introduction

Over the past several decades, drug resistance has emerged as a major obstacle to the treatment and control of diseases of parasitic origin. Leishmania has served as a prototype genus for elucidating the molecular mechanisms and biochemical alterations that provoke drug resistance in parasitic protozoa, due to Correspondence address: Buddy Ullman, Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97201-3098. Tel.: (503) 494 8437 Fax: (503) 494 8393. Internet: [email protected]

Abbreviations: PFGE, pulsed field gel electrophoresis; PFG, pulsed field gel; CHEF, contour-clamped homogenous electric field electrophoresis; IMPDH, IMP dehydrogenase; MPA, mycophenolic acid; MTX, methotrexate; TM, tunicamycin; DFMO, De-c~-difluoromethylornithine.

the facility by which their culture conditions can be manipulated. One mechanism by which Leishmania species acquire resistance to drugs in vitro is by gene, or DNA sequence, amplification [1]. Amplification of target genes has been reported for Leishmania species isolated for their resistance to methotrexate (MTX), 10-propargyl-5,8-dideazafolate (CB3717), tunicamycin (TM), mycophenolic acid (MPA), and DL-~-difluoromethylornithine (DFMO) toxicity [2-6]. Drug-induced DNA amplifications in Leishmania are often organized as extrachromosomal circular molecules [2,4,7-9]. One o f these extrachromosomal DNAs, the H region, is also amplified in unselected laboratory strains of Leishmania

[10-12]. Wilson et al. [5] have recently characterized a strain of Leishmania donovani, MPA100, that

198

has amplified its IMP dehydrogenase (IMPDH) gene copy number approx. 15 fold. The MPA100 cell line was isolated after continuous selection in increasing concentrations of MPA, a potent inhibitor of I M P D H , the penultimate enzyme in guanylate nucleotide synthesis. We now report that MPA100 cells contain a 280-kb extrachromosomal DNA, IMPDH-280, that is not found in wild type L. donovani. Most of the amplified impdh gene copies are located on IMPDH-280. IMPDH-280 was characterized by its electrophoretic behavior in pulsed field gels (PFGs) and shown to be linear in nature. This is the first report of a stable amplification of a linear extrachromosomal D N A in parasites selected for resistance to drugs.

Materials and Methods

Chemicals and reagents. Mycophenolic acid 6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-5phthalanyl)-4-methyl-4-hexenoic acid) was a generous gift from Ronald Davis of the Eli Lilly Corporation (Indianapolis, IN). InCert agarose was purchased from F M C BioProducts (Rockland, ME). All other materials, reagents, and chemicals were of the highest quality commercially available. Cell culture. L. donovani promastigotes were propagated in a completely defined culture medium, DME-L-H, as described by Wilson et al. [5]. DME-L-H is a modification of the completely defined D M E - L growth medium originally reported by Iovannisci and Ullman [13], except that hypoxanthine replaces xanthine as a purine source. Parasites were grown continuously in a humidified 10% CO2 atmosphere at 26°C. The wild type DI700 cell line is a clonal derivative of the 1S Sudanese strain of L. donovani. The selection and phenotypic characterization of the MPA100 L. donovani cells has been reported previously [5]. Except for stability studies, MPA100 cells were perpetually grown in DME-L-H containing 100 #M MPA. The growth inhibitory effects of MPA on MPA100 cells grown in the absence

of MPA were determined as reported [5].

DNA probes and Southern blot hybridization. The impdh probe, pl01, is a 2.3-kb EcoRV-PstI fragment that contains the entire protein coding region of the L. donovani impdh gene [5]. priM3 is a 2.0-kb HindIIl fragment obtained from H circle D N A from MTXresistant Leishmania major [2]. pK25 is a 2.5kb KpnI D N A that encompasses a portion of the ltpgpA gene and was originally cloned from the D N A of an unselected stock of Leishmania tarentolae bearing an H region amplification [11]. The L. donovani multidrug resistance gene, ldmdrl, is present on a 5.7-kb XmaI fragment that was isolated as described by Henderson et al. [14]. A 4.5-kb SacI-SalI fragment encoding the L. donovani ornithine decarboxylase gene has been characterized by Hanson et al. [6]. p7R50-19 is a 1.9-kb PstI fragment obtained from the small linear DNAs found in multiple Leishmania species [15], and pCD-1-B8 is a 3.5-kb BamHI fragment obtained from Leishmania infantum CD1 D N A [16]. pTT6 is a probe to the telomere repeat unit of T. brucei [17]. Southern blot analysis of genomic D N A was performed as described previously [5]. Pulsed field gel electrophoresis and 7-irradiation. 2 x 10 7 DI700 or MPA100 promastigotes were washed twice with phosphate buffered saline (PBS) and resuspended in PBS mixed 1:1 with 1.0% InCert agarose. The cells were lysed in 0.5 M EDTA (pH 8.0), 1 % Sarkosyl, and 2 mg m l - 1 proteinase K at 55°C for 48 h. The agarose blocks were washed 3 times in excess sterile 10 m M Tris (pH 7.4) and 1 m M E D T A and stored at 4°C. Two types of pulsed field gel electrophoresis (PFGE) were performed, the first on a noncommercially available uniform field and gel rotation apparatus [18] and the second on a contourclamped homogeneous electric field (CHEF) apparatus from Bio-Rad (DR-II). Voltage gradients, pulse times, and the duration of the electrophoresis were varied as fitting for the size range of DNAs to be separated. Southern blot analysis of PFGs using alkaline

199 transfer conditions was performed after depurination of the chromosomal DNA in 0.25 M HCI for 30 min. 7-Irradiation of the MPA100 chromosomes in the agarose plugs was performed either in the absence of buffer using a JL Shepard and Assoc. Mark 1, model 68 irradiator employing a 137Cs source or a ICN GR9 6°Co source using the conditions outlined by Beverley [19].

2-Exonuclease

digestion of

IMPDH-280.

MPA100 chromosomes were fractionated by PFGE, and IMPDH-280 was excised from the gel. The agarose plugs containing IMPDH-280 were equilibrated in 2-exonuclease buffer for 1 h at room temperature. 2-exonuclease was then added directly to the plugs at a concentration of 0.05 U #1- l, and the reaction was allowed to proceed for 0, 10, 30, 60, and 120 min at 37°C. 1 pg of a control plasmid, p 101, was incubated with the 2-exonuclease under identical reaction conditions. The 2-exonuclease reactions were terminated by the addition of 5 mM EDTA, and the reaction products were fractionated either by PFGE (IMPDH-280) or on an 0.8% agarose gel (pl01).

Alkaline lysis procedure for plasmid isolation. The alkaline lysis procedure for extracting circular DNA molecules was carried out as described by Davis et al. [20].

Results

Extrachromosomal DNA in MPAIO0 cells. To investigate the nature of the DNA amplification in MPA100 cells, chromosomes were fractionated by PFGE using a 60 s pulse time (Fig. 1A). As demonstrated in the ethidium bromide-stained PFG depicted in Fig. 1A, MPA100 cells contained a conspicuous additional DNA fragment, designated IMPDH-280, that migrated with an apparent size of 280 kb. No IMPDH-280 could be visualized in parental DI700 chromosomes. Southern blot analysis indicated that virtually all of the extra copies of impdh could be localized to IMPDH-280 (Fig. 1B). The impdh

probe also hybridized to 2 chromosomal sized DNAs in MPA100 cells. The first, a 700-kb chromosome, appeared to be the wild type impdh chromosome since the hybridization signals to the DI700 and MPA100 chromosomes were equivalent in size and intensity. The second MPA100 chromosome to which the impdh probe hybridized migrated with a size of 740 kb (Fig. 1B). No hybridization was observed at that Mr with DI700 chromosomes. IMPDH-280 did not appear to exhibit substantial homology to previously characterized amplified leishmanial DNAs as determined by Southern blot analysis of PFGs of DI700 and MPA100 chromosomes. IMPDH280 was not recognized by either priM3 or pK25, 2 DNAs originating from different segments of the H region of L. major and L. tarentolae, respectively, by p7R50-19, a probe derived from one of the small linear DNAs found in L. major [15], or by pCD-1-B8, a probe obtained from the circularized form of LD1 DNA from L. infantum [16]. The L. major small linear DNAs have been shown to be related to LD1 in a complex manner [15]. Finally, no amplification of the leishmanial ornithine decarboxylase gene that is amplified in DFMO-resistant L. donovani [6] or of the ldmdrl multidrug resistance gene that is amplified in multidrug-resistant L. donovani [14] was observed in MPA100 DNA.

Topology of IMPDH-280. To establish whether IMPDH-280 was a circular or linear molecule, the relative mobility of IMPDH280 was compared to that of linear markers as a function of pulse time. The relative migration of IMPDH-280 compared to the linear yeast markers or to the large linear leishmanial chromosomes using a 30-s pulse time was indistinguishable from that observed with a 60s pulse time (Figs. 1A and 1C). The absolute migration of IMPDH-280, however, was dependent on pulse time. The pulse timeindependent relative migration of IMPDH280 in PFGs was also noted for several other pulse times (data not shown). Moreover, IMPDH-280 migrated within the track of the linear chromosomes when MPA100 chromo-

200

A.

B.

o o

C.

o O

n

E3

O O r,-

O o

a.

I~

D..

450

375

295 225

Fig. 1. P F G E of DI700 and M PAI00 chromosomes. PFGs of DI700 and M P A I 0 0 chromosomes using a pulse time of 60 s (A and B) or 30 s (C) were stained with ethidium bromide (A and C). A Southern blot of the P F G depicted in A was probed with the L. donovani impdh gene (B). The arrow in A designates IMPDH-280. The Mrs of leishmanial DNAs were estimated by comparing their mobilities to those of yeast chromosomal markers. The yeast markers were obtained from the S. cerevisiae strain YNN295.

somes were fractionated in a lane located at the edge of a C H E F gel (data not shown). The electrophoretic behavior of I M P D H - 2 8 0 in PFGs, therefore, was characteristic of that of a linear D N A molecule [21]. Another technique to differentiate between circular and linear D N A molecules is to assess their mobilities by P F G E after v-irradiation. Limited v-irradiation linearizes circular D N A molecules to discretely sized linear molecules by introducing a single, random, double-strand break. The sizes of these linearized fragments can then be readily assessed by P F G E [19]. Exposure of MPA100 chromosomes in agarose plugs to 2-9 krad of v-irradiation from a 137Cs source did not affect the migration or cause the disappearance of IMPDH-280, as assessed by both ethidium bromide staining (Fig. 2A) and

hybridization to the impdh gene (Fig. 2B). Similar results were obtained after fractionation of MPA100 chromosomes on a C H E F gel apparatus that had been subjected to 100 krad of v-irradiation from a 6°Co source (data not shown). In addition, no new discrete D N A bands were observed after v-irradiation. Although the experiments in Fig. 2 do not formally exclude the appearance of another small D N A after v-irradiation by the ~37Cs source, the C H E F gel performed on MPA100 chromosomes that were irradiated with the 6°Co source revealed no new discrete D N A s > 10 kb (data not shown). Several other experimental manipulations also suggested that I M P D H - 2 8 0 was linear in nature. First, no circular D N A s were observed in MPA100 cells subjected to the alkaline lysis

201

A.

280 kb -

7

’

280

Fig. 2. y-Irradiation of MPAIOO chromosomes. Agarose plugs containing MPA 100 chromosomes received 2, 6, or 9 krad of y-irradiation from a 13’Cs source as described in Materials and Methods. The irradiated chromosomes were subjected to PFGE for 38 h on a CHEF gel apparatus using a beginning pulse time of 60 s that increased linearly to 120 s over the course of 38 h. The PFG was stained with ethidium bromide (A), blotted to Nytran, and hybridized to, the L. donovani impdh gene (B).

procedure for plasmid isolation (data not shown). A control manipulation of DFMOresistant L. donovani [6] by this protocol resulted in the isolation of large quantities of circular DNA molecules (S. Hanson, personal communication). Second, IMPDH-280 was susceptible to digestion by A-exonuclease under conditions that did not affect the integrity of a control circular plasmid (Fig. 3) or a 70-kb circular extrachromosomal DNA present in DFMO-resistant L. donovani (S. Hanson, personal communication). Therefore, the degradation of IMPDH-280 reflects exonuclease activity rather than endonucleolytic cleavage by a contaminant in the i-exonuclease preparation. These data support the conjecture that IMPDH-280 is a linear molecule. Telomeric sequences on IMPDH-280. To ascertain whether IMPDH-280 contained

B.

0

10

30

60

120

Fig. 3. I-exonuclease digestion of IMPDH-280. IMPDH280 in agarose plugs was digested with I-exonuclease for 0, IO, 30, 60 and 120 min. The digestion products were fractionated on a CHEF gel apparatus for I8 h starting with a pulse time of 45 s that increased in a linear fashion to a final pulse time of 90 s at 18 h. The PFG was stained with ethidium bromide (A). A control 5.3-kb plasmid, ~101, was digested with A-exonuclease under the identical reaction conditions used for IMPDH-280, and the product was fractionated on an 0.8% agarose gel ( B).

telomeric sequences, a PFG of MPAlOO chromosomes was hybridized to pTT6, a probe encoding the telomere repeat unit of T. hybridized readily to the brucei. pTT6 IMPDH-280 amplicon, as well as to the linear chromosomes (Fig. 4). Furthermore, the circular DNA molecule found in DFMOresistant L. donovani (S. Hanson, personal communication) did not hybridize to the pTT6 probe. These data suggest, therefore, that IMPDH-280 contains telomeres.

202

(:3 0

o r.-

remained resistant to MPA after growth for 23 months in the absence of drug.

<1: 13.

Discussion

kb

450

375

295

225

Fig. 4. Presence of telomeric sequences on IMPDH-280. A Southern blot of the P F G depicted in Fig. 1C was hybridized to pTT6, a probe derived from the telomere repeat of T. brucei [17].

Mitotic stability of IMPDH-280. To assess the stability of IMPDH-280, MPA100 cells were removed from MPA-containing medium at various times, and their chromosomes fractionated by PFGE. As shown by the ethidium bromide stained P F G in Fig. 5A, a 280-kb D N A was maintained in MPA100 cells grown in the absence of selective pressure for 7, 17, and 23 months. Southern blots of these PFGs demonstrated that the 280-kb D N A was IMPDH-280 since it hybridized strongly to the leishmanial impdh gene (Fig. 5B). Thus, IMPDH-280 was stable in the absence of selective pressure. The stability of I M P D H 280 within MPA100 cells was correlated with the preservation of its cellular drug resistance phenotype. As shown in Fig. 6, MPA100 cells

The amplification of the impdh gene and the growth phenotype of MPA-resistant L. donovani can be attributed to the presence of a 280-kb linear extrachromosomal DNA, IMPDH-280. The linear topology of IMPDH-280 is supported by its pulse time independent mobility relative to the linear chromosomes and yeast standards, by the failure of limited v-irradiation to alter its migration in PFGs or to generate a new discrete D N A band, by its 'in-track' migration in PFGs under nonhomogeneous electric field conditions, by its hybridization to a parasite telomere repeat probe, and by its sensitivity to 2-exonuclease digestion. The amplification of a linear extrachromosomal D N A is rather atypical of drug-selected Leishmania species that have arisen following a gene amplification event. Previously reported extrachromosomal elements in drug-resistant Leishmania have all been circular molecules. These include the R region in MTX- and CB3717-resistant Leishmania that encompasses the dihydrofolate reductase-thymidylate synthase gene [2,3,22], the H region in Leishmania selected for their resistance to MTX, arsenite, primaquine, or terbinafine that contains a gene encoding a member of the P-glycoprotein family [2,7,8,23], and a circular amplicon observed in TM-selected Leishmania that overexpress N-acetylglucosamine- 1-phosphate transferase activity [4]. Recently, however, a linear 140-kb amplified D N A has been identified in DFMO-resistant L. donovani (S. Hanson, personal communication). Finally, it should be noted that a variety of multicopy D N A molecules, both circular [10,16,24] and linear [15], with no apparent connection to drug resistance, have been observed in several Leishmania species. IMPDH-280 is stable in the absence of selective pressure for over 23 months. Thus, IMPDH-280 appears to contain the necessary

203

A.

MPAl 00

B.

MPAl 00

61-h::

[r,,,,i

-kb

kb 740 700 -

Fig. 5. Stability of IMPDH-280 in the absence of selective pressure. PFGE was performed on chromosomes from MPA 100 cells continuously cultivated in 100 PM MPA and on chromosomes from cells removed from selective pressure for either 7, 17 or 23 months. PFGE was performed under conditions identical to those described in the legend to Fig. 2. The PFG was stained with ethidium bromide (A), blotted to a Nytran filter, and probed with the L. donovani imp& gene (B).

Mf’A (PM)

Fig. 6. Stability of the drug resistance phenotype in the absence of selective pressure. The growth inhibitory effects of MPA on D1700 (a), MPAIOO (O), and MPAIOO cells propagated in the absence of MPA for 7 (A), 17 (U), and 23 (0) months were determined as described in the ‘Materials and Methods’ section. _

c&acting elements mediating mitotic stability. How these potential centromeric sequences are arranged in Leishmania DNA has not been elucidated. The existence of telomeric sequences, presumably at the termini of the linear 280-kb DNA, can account for the faithful replication and the mitotic stability of IMPDH-280. The stability of the 280-kb extrachromosome in MPAlOO cells is accompanied by a stable drug resistance phenotype. In contrast to IMPDH-280, the linear extrachromsomal DNA detected in DFMO-resistant L. donovani [6] is unstable in the absence of selective pressure. Thus, the linearity of IMPDH-280 is not sufficient to confer the stability of the extrachromosome. The advent of transfection systems in Leishmania [25,26] affords the opportunity to genetically dissect those determinants required for ensuring

204

chromosomal stability in these parasites. In comparison to the linear amplified DNAs, the circular extrachromosomal DNAs that have been detected in Leishmania can either be unstable [2,12,27] or stable [2,22]. Initially, the copy number of amplified circular DNAs in drug-resistant Leishmania decreases rapidly after removal of the parasites from selective pressure with a concomitant restoration of the wild type growth phenotype. After prolonged propagation in the presence of the drug, however, the growth phenotype and the circular amplified DNAs become stabilized [2] although this stability, unlike that observed in stably amplified mammalian DNAs, does not appear to involve a chromosomal relocalization event [22]. Interestingly, there appears to be an extra copy of the impdh gene in MPA100 cells that is found on a 740-kb chromosome. There are several models that could account for this observation. For instance, it is possible that sequences encompassing the impdh gene on IMPDH-280 reintegrated into the chromosomal DNA. Alternatively, and also consistent with restriction maps of the impdh locus in DI700 [5] and MPA100 D N A (data not shown), is the interpretation that the 700-kb and the 740-kb chromosomes are homologous chromosomes which have generated a size polymorphism during or subsequent to the process of selection of the MPA100 strain from the DI700 parental stock. Polymorphisms of 40-50 kb in homologous chromosomes, affecting up to 20% of the chromosome length, have been documented in Leishmania [28 30], as well as in T. brucei [31 33]. Several karyotypic differences can also be discerned between the parental DI700 and MPA100 chromosomes (Figs. 1 and 5). Although incompletely understood, chromosomal plasticity has been documented within the genomes of Trypanosoma [31] and Leishmania [34,35] species, including cell lines undergoing drug selection [30]. For instance, Rovai et al. [36] have observed chromosomal size polymorphisms in L. tarentolae induced either by nutrient stress or by single cell cloning. It is conceivable that the polymorph-

isms observed between DI700 and MPAI00 chromosomes might have been provoked or influenced by a diminution of the guanylate nucleotide pool by continuous exposure to the I M P D H inhibitor. Thus, chromosomal polymorphisms may be a general phenomenon in Leishmania undergoing stress, including drug selection. The appearance of a linear extrachromosomal D N A related to drug resistance suggests 2 possible models for its de novo formation. Either, IMPDH-280 originates from the middle of a chromosome and requires the addition of 2 telomeres or is generated from the end of a chromosome followed by the attachment of a single telomere. These models can be differentiated by physical mapping of IMPDH-280 and its chromosomal counterpart. The origin of the IMPDH-280 amplicon is likely to be a consequence of a conservative rather than a deletional amplification, although a duplicative amplification mechanism cannot be ruled out due to the presence of the 740-kb chromosome [1].

Acknowledgements This work was supported by Grants AI23682 and AI32036 (B.U.) and Grant AI21903 (S.B) from the National Institute of Allergy and Infectious Diseases. K.W.'s salary was supported in part by the N. L. Tartar Trust Fellowship Fund. We thank C. M. Coburn, D. D. Rogers and L. Tolmasky for technical assistance and Dr. Kenneth Stuart and Dr. Lex van der Ploeg for providing the pCD-1-B8 and pTT6 probes, respectively. We also thank Dr. Sheri Hanson for critical reading of the manuscript and many helpful discussions.

References 1 Beverley, S.M. (1991) G e n e a m p l i f i c a t i o n in Leishmania. Annu. Rev. Microbiol. 45, 417-444. 2 Beverley, S.M., Coderre, J.A., Santi, D.V. and Schimke, R.T. (1984). Unstable D N A amplifications in methotrexate-resistant Leishmania consist of extrachromosomal circles which relocalize during stabilization. Cell 38,

205 431-439. 3 Garvey, E.P., Coderre, J.A. and Santi, D.V. (1985) Selection and properties of Leishmania tropica resistant to 10-propargyl-5,8-dideazafolate, an inhibitor of thymidylate synthetase. Mol. Biochem. Parasitol. 17, 79-91. 4 Katakura, K., Peng, Y., Pithawalla, R., Detke, S. and Chang, K.P. (1991) Tunicamycin-resistant variants from five speicies of Leishmania contain amplified DNA in extrachromosomal circles of different sizes with a transcriptionally active homologous region. Mol. Biochem. Parasitol. 44, 233-244. 5 Wilson, K., Collart, F.R., Huberman, E., Stringer, J.R. and Ullman, B. (1991) Amplification and molecular cloning of the IMP dehydrogenase gene of Leishmania donovani. J. Biol. Chem. 266, 1665 1671. 6 Hanson, S., Adelman, J. and Ullman, B. (1992) Amplification and molecular cloning of the ornithine decarboxylase gene from Leishmania donovani. J. Biol. Chem. 267, 235~2359. 7 Ouellette, M., Fase-Fowler, F. and Borst, P. (1990) The amplified H circle of methotrexate-resistant Leishmania tarentolae contains a novel P-glycoprotein gene. EMBO J. 9, 1027 1033. 8 Ellenberger, T.E. and Beverley, S.M. (1989) Multiple drug resistance and conservative amplification of the H region in Leishmania major. J. Biol. Chem. 264, 15094~ 15103. 9 Katakura, K. and Chang, K.P. (1989) H DNA amplification in Leishmania resistant to both arsenite and methotrexate. Mol. Biochem. Parasitol. 34, 189192. 10 Hightower, R.C., Ruiz-Perez, L.M., Wong, M.E. and Santi, D.V. (1988) Extrachromosomal elements in the lower eukaryote Leishmania. J. Biol. Chem. 263, 16970 16976. 11 Petrillo-Peixoto, M.L. and Beverley, S.M. (1988) Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted H-region amplification of methotrexate-resistant Leishmania major. Mol. Cell Biol. 8, 5188-5199. 12 White, T.C., Fase-Fowler, F., van Luenen, H., Calafat, J. and Borst, P. (1988) The H circles of Leishmania tarentolae are a unique amplifiable system of oligomeric DNAs associated with drug resistance. J. Biol. Chem. 263, 16977 16983. 13 Iovannisci, D.M. and Ullman, B. (1983) High efficiency plating method for Leishmania promastigotes in semidefined or completely defined medium. J. Parasitol. 169, 633436. 14 Henderson, D.M., Sifri, C.D., Rodgers, M., Wirth, D.F. and Ullman, B. (1992) Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdrl gene. Mol. Cell Biol. 12, 2855-2865. 15 Beverley, S.M. and Coburn, C. (1990) Recurrent de novo appearance of small linear DNAs in Leishmania major and relationship to extra-chromosomal DNAs in other species. Mol. Biochem. Parasitol. 42, 133-142. 16 Tripp, C., Myler, P. and Stuart, K. (1991) A DNA sequence (LDI) which occurs in several genomic organizations in Leishmania. Mol. Biochem. Parasitol. 47, 151-160. 17 Van der Ploeg, L.H.T., Lui, A.Y.C. and Borst, P. (1984)

The structure of the growing telomeres of trypanosomes. Cell 36, 459-468. 18 Anarand, R. (1986) Pulsed field gel electrophoresis: a technique for fractionating large DNA molecules. Trends Genet. 2, 278-283. 19 Beverley, S.M. (1989) Estimation of circular DNA size using y-irradiation and pulsed-field gel electrophoresis. Anal. Biochem. 177, 110 114. 20 Davis, L.G., Dibner, M.D. and Battey, J.F. (1986) Basic Methods in Molecular Biology. Elsevier, New York. 21 Beverley, S.M. (1988) Characterization of the 'unusual' mobility of large circular DNAs in pulsed field gradient electrophoresis. Nucleic Acids Res. 16, 925 939. 22 Garvey, E.P. and Santi, D.V. (1986) Stable amplified DNA in drug resistant Leishmania exists as extrachromosomal circles. Science 233, 535 540. 23 Callahan, H. and Beverley, S.M. (1991) Heavy metal resistance: a new role for P-glycoproteins in Leishmania. J. Biol. Chem. 266, 18427 18430. 24 Petrillo-Peixoto, M.L. and Beverley, S.M. (1989) Amplification of a new region of DNA in an unselected laboratory stock of Leishmania tarentolae: the T region. J. Protozool. 36, 257 293. 25 Lebowitz, J.H., Coburn, C.M., McMahon-Pratt, D. and Beverley, S.M. (1990) Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes. Proc. Natl. Acad. Sci. USA 87, 9736 9740. 26 Laban, A., Tobin, J.F., Curotto de Lafaille, M.A. and Wirth, D.F. (1990) Stable expression of the bacterial neo r gene in Leishmania enriettii. Nature 343, 572 574. 27 Kink, J.A. and Chang, K.P. (1987) Tunicamycinresistant Leishmania mexicana amazonensis: Expression of virulence associated with an increased activity of Nacetylglucosaminyltransferase and amplification of its presumtive gene. Proc. Natl. Acad. Sci. 84, 1253 1257. 28 Blaineau, C., Bastien, P., Rioux, J.A., Roizes, G. and Pages, M. (1991) Long-range restriction maps of sizevariable homologous chromosomes in Leishmania infantum. Mol. Biochem. Parasitol. 46, 293 302. 29 Scholler, J.K., Reed, S.G. and Stuart, K. (1986) Molecular karyotype of species and subspecies o1 Leishmania. Mol. Biochem. Parasitol. 20, 279-293. 30 Iovannisci, D. and Beverley, S.M. (1989) Structural alterations of chromosome 2 in Leishmania major as evidence for diploidy, including spontaneous amplification of the mini-exon array. Mol. Biochem. Parasitol. 34, 177-188. 31 Van der Ploeg, L.H.T., Cornelissen, A.W.C.A., Michaels, P.A.M., Borst, P. (1984) Chromosome rearrangements in Trypanosome brucei. Cell 39, 213 221. 32 Gottesdiener, K., Garcia-Anoveros, J., Lee, M.G.S and Van der Ploeg, L.H.T. (1990) Chromosome organization of the protozoan Trypanosoma brucei. Mol. Cell. Biol. 10, 6079 6083. 33 Gibson, W., Garside, L. and Bailey, M. (1992) Trisomy and chromosome size changes in hybrid trypanosomes from a genetic cross between Trypanosoma brucei rhodesiense and T. b. brucei. Mol. Biol. Parasitol. 52, 189 200. 34 Bishop, R.P. and Miles, M.A. (1987) Chromosome sized polymorphisms of Leishmania donovani. Mol. Biochem. Parasitol. 24, 263 272.

206 35 Bishop, R.P. 0990) Extensive homologies beween Leishmania donovani chromosomes of markedly different size. Mol. Biochem. Parasitol. 38, l 12. 36 Rovai, L., Tripp, C., Stuart, K. and Simpson, L. (1992)

Recurrent polymorphisms in small chromosomes of Leishmania tarentolae after nutrient stress or subcloning. Mol. Biochem. Parasitol, 50, 115 126.