Shuttle mutagenesis and targeted disruption of a

telomere-located essential gene of Leishmania

F. M. SQUINA, A. L. PEDROSA#, V. S. NUNES, A. K. CRUZ and L. R. O. TOSI*

Departamento de Biologia Celular e Molecular e Bioagentes Patogenicos, Faculdade de Medicina de Ribeirao Preto,Universidade de Sao Paulo, Brasil

(Received 19 September 2006; revised 6 October 2006; accepted 10 October 2006; first published online 15 December 2006)

SUMMARY

Leishmaniamutants have contributed greatly to extend our knowledge of this parasite’s biology. Here we report the use of

the mariner in vitro transposition system as a source of reagents for shuttle mutagenesis and targeted disruption of

Leishmania genes. The locus-specific integration was achieved by the disruption of the subtelomeric gene encoding aDNA-

directed RNA polymerase III subunit (RPC2). Further inactivation of RPC2 alleles required the complementation of the

intact gene, which was transfected in an episomal context. However, attempts to generate a RPC2 chromosomal null

mutant resulted in genomic rearrangements that maintained copies of the intact locus in the genome. The maintenance of

the RPC2 chromosomal locus in complemented mutants was not mediated by an increase in the number of copies and did

not involve chromosomal translocations, which are the typical characteristics of the genomic plasticity of this parasite.

Unlike the endogenous locus, the selectable marker used to disrupt RPC2 did not display a tendency to remain in its

chromosomal location but was targeted into supernumerary episomal molecules.

Key words: Leishmania, in vitro transposition, targeted mutagenesis, telomere.

INTRODUCTION

Pathogenic Leishmania are protozoan parasites re-

sponsible for a spectrum of human diseases collec-

tively called leishmaniases (Pearson and Sousa,

1996). The wide range of pathologies depends not

only on the infecting species but also on a complex

host-parasite interaction. The availability of the

Leishmania major genome sequence has provided a

unique insight into the content and organization of

the parasite’s genetic information (Ivens et al. 2005).

Such a comprehensive view of its genome and our

ability to genetically manipulate this trypanosomatid

constitute a fertile ground for the development of

functional studies, such as the generation of mutant

collections of the parasite.

Leishmania is mostly diploid and is believed to

have an asexual life-cycle (Iovannisci et al. 1984).

These features pose a major challenge to the gener-

ation of homozygous mutants, especially when

dealing with phenotypes that cannot be easily

rescued. Attempts to inactivate genes that are es-

sential for this parasite invariably induce DNA

amplification and/or changes in ploidy, revealing a

significant genome plasticity (Cruz et al. 1993;

Dumas et al. 1997). Targeted gene replacement is

widely used to obtain Leishmaniamutants (Cruz and

Beverley, 1990) and may involve several cloning

steps to produce the adequate reagents. Moreover,

the generation of null mutants requires consecutive

rounds of targeting with independent selectable

markers (Cruz et al. 1993; Dumas et al. 1997; Zhang

et al. 2003). Alternatively, loss of heterozygosity

(LOH), which was described for a few loci (Gueiros-

Filho and Beverley, 1996; Pedrosa and Cruz, 2002),

can be achieved after subjecting heterozygous cell

lines of the parasite to increased concentrations of

their selective antibiotic.

Transposon-based mutagenesis constitutes a use-

ful approach not only to the introduction and/or the

inactivation of phenotypes, but also to the study of

the relationship between gene structure and function

(Hamer et al. 2001b). A productive transposition

event can be generated either within the target or-

ganism or by shuttle mutagenesis. In the latter case,

the transposon is mobilized in vivo or in vitro and

then introduced into the target organism aiming at a

phenotypic interference (Ross-Macdonald et al.

1999; Hamer et al. 2001a).

The Drosophila mariner Mos1 transposon belongs

to the Tc1/mariner superfamily of transposable

elements, which are effective instruments for gene-

delivery strategies (Plasterk et al. 1999; Coates et al.

2000; Williams et al. 2005). The malleability of

the Mos1 element allowed the establishment of an

* Corresponding author: Departamento de BiologiaCelular e Molecular e Bioagentes Patogenicos, FaculdadedeMedicina deRibeirao Preto,Universidade de Sao Paulo,Av. Bandeirantes, 3900, 14049-900, Ribeirao Preto – SP,Brasil. Tel : +55 16 3602 3117. Fax: +55 16 633 1786.E-mail : luiztosi@fmrp.usp.br# Current address: Departamento de Ciencias Biologicas,Universidade Federal do TrianguloMineiro, Avenida FreiPaulino, 30, 38025-5045, Uberaba – MG, Brasil.

511

Parasitology (2007), 134, 511–522. f 2006 Cambridge University Press

doi:10.1017/S0031182006001892 Printed in the United Kingdom

in vitro transposition system (Tosi and Beverley,

2000) and the design of a toolkit for shuttle muta-

genesis in this parasite (Goyard et al. 2001; Robinson

et al. 2004). Modified Mos1 elements are a reliable

tool for gene trapping, primer-island sequencing

approaches (Pedrosa et al. 2001; Augusto et al.

2004) and shuttle mutagenesis protocols (Marchini

et al. 2003). This transposon can also mobilize

in vivo within Leishmania (Gueiros-Filho and

Beverley, 1997) and Trypanosoma genomes (Leal

et al. 2004).

Herein we report the use of in vitro shuttle muta-

genesis as a source of reagents for targeted disruption

of the putative second-largest subunit of RNA

Polymerase III (RPC2), a telomere-located

Leishmania gene. The specific integration of the

disruption reagent indicates that this approach is an

efficient and rapid means of producing mutant

parasites. Moreover, the results presented here re-

veal new features of this parasite’s ability to promote

genome rearrangements during the targeting of

an essential gene. The functional complementation

of the targeted gene allowed the further inactivation

of alleles and generated a wide range of reagents that

are adequate for the study of the mechanisms leading

to the observed genome plasticity. The preservation

of RPC2 was not mediated by an increase in number

of copies or by ectopic insertion into other chromo-

somes, which suggests that the genomic plasticity of

this parasite is not restricted to the current models

of DNA rearrangement and amplification (Beverley,

1991; Genest et al. 2005).

MATERIALS AND METHODS

Leishmania cultures and transfection

Promastigote forms ofL.majorLT252 (MHOM/IR/

1983/IR) were grown at 26 xC in M199 medium

supplemented with 10% heat-inactivated fetal calf

serum (Kapler et al. 1990). Transfectants from CC1,

a LT252 clonal line, were selected on M199-agar

plates with 32 mg.mlx1 of hygromycin B (Gibco

BRL) and/or 40 mg.mlx1 of nourseothricin (Werner

Bioagents) as required. Individual clones were

transferred to liquid M199 medium supplemented

with 16 mg.mlx1 of hygromycin B, and/or 20 mg.mlx1

of nourseothricin. Promastigote cells (8.106.mlx1)

were pelleted at 2000 g for 10 min at 4 xC for the

preparation of genomicDNA or RNA extraction and

transfection assays. The electroporation protocol

used has been previously described (Kapler et al.

1990).Mutant parasites were named according to the

nomenclature of Clayton et al. (1998).

DNA and RNA manipulation

Large-scale plasmid or cosmid DNA preparation

was carried out using alkaline lysis followed by

caesium chloride gradient (Sambrook et al. 1989).

Total DNA from promastigotes was extracted as

previously described (Tosi et al. 1997). Bal31 assays

used 2U of the enzyme (Biolabs) for different incu-

bation periods; the reaction was stopped by adding

EGTA to a final concentration of 20 mM and samples

were digested with the restriction enzyme of choice.

Intact chromosomes were prepared in agarose blocks

(Cruz and Beverley, 1990) and resolved by Pulse

Field Gel Eletrophoresis (PFGE) using a Bio-Rad

CHEF Mapper apparatus. The PFGE separation of

episomal DNA used 10-sec constant pulses for 18 h.

Total RNA from promastigotes was extracted using

TRIzol1 (Gibco BRL). Aliquots containing 15 mg oftotal RNA were equilibrated in formamide, formal-

dehyde andMOPS buffer, heated for 15 min at 65 xC

and separated in a 1.5% agarose formaldehyde/

MOPS gel (Sambrook et al. 1989). DNA and RNA

transfer to nylon membranes, hybridization and

washing conditions were conducted as previously

described (Tosi et al. 1997). Radio-isotope labelled

probes were prepared using the random priming

method (Feinberg and Vogelstein, 1983).

Membranes were exposed to a Kodak Diagnostic

Film or to a Molecular Dynamics Storage Phosphor

Screen for densitometric analysis. Polymerase Chain

Reaction (PCR) and Reverse transcriptase-PCR

(RT-PCR) reactions were performed with Ready-

To-Go@ PCR and RT-PCR Beads (Amersham

Pharmacia Biotech) according to the manufacturer’s

specifications using the primers (a) 5k-TAGAAGT-

CGCGCTCGTCAA-3k ; (b) 5k-TTCCTCGCCA-

ACGTGCTACT-3k ; (c) 5k-GAACGCGTGGAT-

CCTCTAG-3k ; (d) 5k-TAGTGCCCACTTTTT-

CGCAA-3k ; (e) 5k-TTGCTATGGCGCATTCAT-

C-3k ; (f) 5k-TGCCCTGTAGCTCTTGGATAA-3kand (g) 5k-TCTCATCAGCTCCGACATGTT-3k.

Molecular constructs

Escherichia coli strains DH10B (Gibco BRL) and

DH5a-lpir (Garraway et al. 1997) were used in this

study. All strains were grown in LB medium sup-

plemented with the appropriate drug (100 mg.mlx1

ampicillin (Gibco BRL); 50 mg.mlx1 hygromycin B;

or 50 mg.mlx1 nourseothricin). Bacteria were

transformed by electroporation or heat-shock of

Ca2+-prepared cells.

The target for transposition was a 4.3 kb HindIII

fragment (HH4.3) subcloned from cosmid E08

(GeneBank Accession number AH010590) (Pedrosa

et al. 2001), which bears 36 kb of the chromosome

20 subtelomeric end. The construction of transposon

ELSAT (1020 bp) included the PCR amplification of

the SAT cassette (the AG sequence, the EM7 pro-

moter and the streptotrycin acetyl transferase gene)

using the primers LT22 (5k-CGCTCTAGAGGA-

TCCAGGCGTTCGAA-3k) and LT23 (5k-CG-

CAGATCTAGATCTCCGAGGCCTG-3k) and

F. M. Squina and others 512

pELSAT as template (Garraway et al. 1997). The

PCR product was digested with XbaI and inserted

into the XbaI site of pELHY6D-0 (Garraway et al.

1997). The plasmid or cosmid DNA templates

bearing the transposon insertion were prepared using

a modified alkaline lysis method (Sambrook et al.

1989) in a 96-well format, followed by a purification

step with 96-well MultiScreen filter plates (Milli-

pore). Sequencing reactions used the Big Dye ter-

minator chemistry with a specific primer for each

transposon. The primers for ELSAT primer island

sequencing were 5k-GAACGCGTGGATCCT-

CTAG-3k (LT25). Single-pass sequencing was car-

ried out on ABI3100 sequencing machine (Applied

Biosystems).

Transposase purification and transposition assay

The recombinant Mos1 transposase preparation

and the transposition reactions were performed as

previously described (Tosi and Beverley, 2000).

The reaction products were recovered following

transformation into E. coli which lacked the pir gene

product.

RESULTS

Shuttle mutagenesis

The target used for disruption by shuttle mutagen-

esis is presented in Fig. 1A. The RPC2 gene

(LmjF20.0010) is the nearest to one of the telomeric

ends of L. major (LT252) chromosome 20 (Pedrosa

et al. 2001). As shown in the figure, the fragment

HH4.3 used as target for transposition contains 92%

of the predicted RPC2. Sequencing of cosmid E08

confirmed that the RPC2 gene did not present any

noticeable alteration, as revealed by sequence align-

ment of the episomal and genomic copies of the gene

(data not shown).

RV A

RP1RP2

A

TEL

4 Kb

0.5 K b

B BNRV K HSH

////a b

H01

Sc SpP

f g

B

H01^rpc2::SAT

RPC2

SATR

AG3' IR 5' IR

d ec

SAT

200 bps

H RV

Fig. 1. Shuttle mutagenesis of the telomere-located RPC2 gene. (A) Schematic representation of Leishmania major

chromosome 20 extremity contained in cosmid E08. As indicated, the 4.3 kb HindIII fragment was used as target for in

vitro transposition. Vertical lines across RPC2 gene represent mapped insertions irrespective of the direction of the

transposon. The event of interest H01 is marked with a closed triangle. RP1 and RP2 represent the probes used in

Southern and Northern experiments; arrows a, b, f and g (out of scale) indicate location and direction of primers used

in PCR or RT-PCR protocols ; H, HindIII; Sp, SphI; Sc, SacI; B, BstEII; N, NarI; E, EcoRV; P, PvuII; S, SalI;

K, KpnI. (B) Schematic representation of the disruption fragment H01^rpc2 : :SAT containing the modified Mos1

transposon ELSAT. The grey arrows represent the selectable resistance marker streptothricin acetyl transferase gene

(SATR); 5kIR, mariner 5k inverted repeat ; 3kIR, mariner 3k inverted repeat ; AG, Leishmania trans-splicing acceptor site;

arrowheads upstream of resistance genes represent Escherichia coli promoter sequence; SAT represents the probe used

in Southern and Northern experiments; arrows c, d and e (out of scale) indicate location and direction of primers used

in PCR or RT-PCR protocols.

Shuttle mutagenesis and targeted disruption of Leishmania genes 513

The transposon used in the disruption protocol is

shown in Fig. 1B. The construction of transposon

ELSAT and its potential application in disruption

strategies have been described elsewhere (Goyard

et al. 2001). The mobile element ELSAT contains a

shuttle cassette encoding the streptotrycin acetyl

transferase gene (SAT). The efficiency of in vitro

transposition of ELSAT into different targets is

comparable to that of modified Mos1 transposons

(Tosi and Beverley, 2000). The insertion events

across fragment HH4.3 are indicated in Fig. 1A and

were identified by restriction mapping and/or primer

island sequencing. As previously reported for other

Mos1 transposons (Augusto et al. 2004), the inser-

tion of these elements showed little regional speci-

ficity or orientation bias (P>0.05; x2 test). The

selected insertion event used to generate the linear

fragment for target disruption of RPC2 is indicated

in Fig. 1A as H01. The major features of the dis-

ruption fragment H01^rpc2::SAT are shown in

Fig. 1B. Varying amounts of the fragment, which

contains the selectable marker flanked by 0.8 and

1.2 kb of the target sequence, were transfected into

the parasite.

Targeted disruption of RPC2 gene

A wild-type cell line (L. major LT252) was trans-

fected with the targeting fragmentH01^rpc2 :SAT to

generate the disruption of a single RPC2 allele.

Clones were selected in medium containing nour-

seothricin. Two transfection experiments using 2

and 5 mg of the gel-purified targeting fragment re-

sulted in the selection of 2 and 4 clones, respectively.

The targeted disruption was confirmed by Southern

analysis of EcoRV-digested genomic DNA from

these 6 clones using fragment RP1, RP2 and SAT

probes (probe location is depicted in Fig. 1A and B).

Fig. 2A shows that probe RP1 revealed the expected

4.3 kb fragment from the disrupted gene and a wild-

type 3.3 kb fragment from the intact loci for clones 1

and 2. The SAT probe detected the 4.3 kb in both

clones. The use of probe RP2, which detected not

only the EcoRV fragment bearing the integration

but also the telomere-proximal EcoRV fragment,

suggested the overall integrity of the locus after tar-

geted disruption (Fig. 2A). Hybridization of PFGE-

separated chromosomal DNA revealed the presence

of the SAT marker on a y760 kb chromosome in

B

A

RP2 SAT_

_

_

2.3 _

6.5 _

4.3 _

2.3 _

_

_

RP1 _

_

_

_

_

_

_

_

_

2.3 _

_

_

2.3 _

_

_

WT 1 2 WT 1 2

_

_

_

_

_

_

WT 1 2

_

_

_

_

0.4_

0.8_

WT 1 2 WT 1 2 WT 1 2

_

_

_

_

_

_

AT

C

_

_

_

_

_

RP1_

_

_

_

_

4.3

9.4

6.5

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_

_4.3

9.4

6.5

0 1h 2h 3h

*

SAT

D

WT

a/b a/c d/e f/g

HZ.2

a/b a/c d/e f/g

RP1 S

Fig. 2. Specific integration of the fragment H01^RPC2: SAT into the genome. (A) Southern experiments from the

wild-type (WT) and mutant clones HZ.1 (1) and HZ.2 (2), derived from 2 distinct transfection experiments of the

targeting fragment; Southern blots of EcoRV-digested genomic DNA were probed with fragments RP1, RP2 and SAT

(refer to Fig. 1). (B) The same probes were used in blots of PFG-separated chromosomes from parental and mutant cell

lines. (C) Time-course digestion of clone HZ.2 genomic DNA with exonuclease Bal31; samples were further digested

with SalI and analysed in Southern blots; signal of a 260-nt internal probe, located downstream of the DHFR-TS gene

in chromosome 6, is marked with an asterisk and confirmed the specificity Bal31 activity. (D) The targeted disruption

of the RPC2 was further confirmed by PCR of WT and HZ.2 genomic DNA using different sets of primers (refer to

Fig. 1 for location of primers a, b, c, d, e, f and g); the arrowhead and the arrow indicate the amplification of 1160 and

160 bp-sized fragments, respectively.

F. M. Squina and others 514

the selected heterozygous clones (Fig. 2B). In ad-

dition, the RP1 probe detected the equivalent chro-

mosomal band in the wild-type cell line and in clones

HZ.1 and HZ.2 (RPC2/^rpc2:: SAT.1 and .2). The

specific integration of the disrupted locus was further

confirmed in Bal31 nuclease assays. As shown in

Fig. 2C, the exonuclease activity on genomic DNA

from mutant HZ.2 caused a progressive shortening

of theSalI fragment, which bears theRPC2 gene and

the SAT marker at one of the chromosomal ends.

The use of an internal probe, which detects a 260-nt

stretch downstream of the DHFR-TS gene in chro-

mosome 6, confirmed the specificity of the Bal31

nuclease activity and is indicated with an asterisk in

Fig. 2C. The targeted disruption of RPC2 was also

demonstrated using PCR with different primer sets

(presented in Fig. 1A) and cloneHZ.2 genomicDNA

as a template. Amplification of truncated RPC2

(primer sets a/b and a/c) and SAT (primer set d/e)

was only detected in the disrupted cell line (Fig. 2D).

The integration of the SAT marker in the genome of

selected mutants was maintained even after removal

of nourseothricin selective pressure for more than

300 generations (data not shown). Table 1 sum-

marizes the characteristics of clone HZ and specific

mutants used in this work.

Further inactivation of RPC2 copies

The generation of a RPC2 null mutant was at-

tempted by inducing the LOH, an approach pre-

viously used in Leishmania (Gueiros-Filho and

Beverley, 1996; Pedrosa and Cruz, 2002). The het-

erozygous cell line was cultured in 400 mg.mlx1

of nourseothricin in order to disrupt other copies of

RPC2. This strategy did not lead to the generation of

aRPC2 null mutant, which indicates thatRPC2 is an

essential gene. Southern analysis of EcoRV-digested

genomic DNA from two selected clones revealed the

preservation of an intact RPC2 locus (Fig. 3A).

Hybridization of PFGE-separated chromosomal

DNA from these clones also showed an amplification

of the SAT marker. As seen in Fig. 3B, one of

the selected clones (HZ.1400) carried an amplified

SAT-containing episome, while the other (HZ.2400)

seemed to present an altered ploidy.

As described for another Leishmania gene (Tovar

et al. 1998), we expected chromosomal null mutants

of the RPC2 gene to be generated after functional

complementation. Therefore, the cosmid bearing the

intact RPC2 gene was transfected into the hetero-

zygous cell line (HZ.1 clone shown in Fig. 2).

Table 1. Summary of mutant Leishmania major clones

Clone Characteristics Selection regime Figure

HZ.1 Single disruption of RPC2 gene nourseothricin 20 mg.mlx1 Figs 2; 4 and 6HZ.2HZ.1400 Derived from HZ. after selection

in high nourseothricin dose.nourseothricin 400 mg.mlx1 Fig. 3

HZ.2400

HZ400[E0816] Derived from HZ.1 after transfectionof cosmid E08 and selection in highnourseothricin dose.

nourseothricin 400 mg.mlx1

hygromycin B 16 mg.mlx1Fig. 4; lanesHZ400[E0816]

HZ0 Derived from HZ400[E0816] cultivatedin the absence of drug pressure for200 generations.

None Fig. 4; lanes HZ0

HZ[E0816] Derived from HZ aftertransfection of cosmid E08.

hygromycin B 16 mg.mlx1 Fig. 4 and 6; lane 1

LOH[E0816] Same as HZ[E0816] ; bears disruptionof all chromosomal copies of RPC2.

hygromycin B 16 mg.mlx1 Fig. 6; lane 2

0.8_

1.4_

0.4_

21B

2.3 _

6.5 _

4.3 _

WT 21A

21

Fig. 3. Attempt to generate a RPC2 null mutant.

Southern analysis of EcoRV-digested genomic DNA (A)

and PFG-separated chromosomes (B) of parental cell line

(WT) and mutants HZ.1400 and HZ.2400 (1 and 2,

respectively), which were selected after culturing the

heterozygous cell line in 400 mg.mlx1 nourseothricin.

Southern blots were probed with fragment RP1.

Shuttle mutagenesis and targeted disruption of Leishmania genes 515

Following transfection of cosmid E08, selected

clones were cultured in 400 mg.mlx1 of nourseo-

thricin for 100 generations. Cell lines selected under

this condition were named HZ400[E0816]. The re-

sulting disruption events were tested using Southern

analysis of KpnI-digested genomic DNA. The en-

donuclease KpnI cleaves upstream of RPC2 and re-

leases the end of chromosome 20 (Fig. 1A). This

restriction pattern allows the discrimination between

the episomal and the wild-type chromosomal RPC2

loci (y20 kb and y9.7 kb fragments, respectively).

As seen in Fig. 4A, Southern analysis using RP1 and

SAT probes revealed that the heterozygous mutant

HZ bears at least 2 intact copies ofRPC2, in addition

to the targeted copy. These results suggest the

existence of at least 3 copies of this gene in the par-

ental cell line LT252. The distinction among these

copies was not possible in regular electrophoresis

conditions, as seen in Fig. 2C, and required a dif-

ferent electrophoresis protocol, as indicated in Fig. 4

legend.

Southern analysis ofHZ400[E0816] clones using the

RP1 probe detected KpnI fragments that varied in

size from y7 to more than 20 kb, in contrast to the

wild-type cell line. Such fragments, indicated by

open circles in Fig. 4A and B, were not detected by

the SAT probe, which suggests they were un-

disrupted copies ofRPC2.Moreover, the intensity of

the hybridization signals of these new KpnI frag-

ments does not indicate DNA amplification of the

locus. The preservation of undisrupted chromo-

somal copies of RPC2 in HZ400 [E0816] clones was

also confirmed by the loss of cosmid E08 in the

absence of hygromycin B selective pressure. As seen

in Fig. 4, Southern analysis of PFG-separated

chromosomes and KpnI-digested genomic DNA

from hygromycin B-sensitive (HZ0 clones) with RP1

and SAT probes revealed the loss of the episomal

molecule, which is indicated by an asterisk in Fig.

4A, B and C. It is noteworthy that the fragments of

different sizes bearing intact copies of RPC2 were

maintained in HZ0 clones. In spite of the size

A

RP1 SAT

-

-

-

-

-

-

23.2

9.4

~7.0

*

WT HZ 1 2 3 4 5 6 1 2 3 4 5 6 7WT HZ 1 2 3 4 5 6 1 2 3 4 5 6 7

HZ400[E0816] HZ0 HZ400[E0816] HZ0B

C

_

_

_

WT HZ 2 3 1 3 6 7

HZ0

HZ400

[E0816]

WT HZ 2 3 1 3 6 7

HZ0

HZ400

[E0816]

WT HZ 2 3 1 3 6 7

HZ0

HZ400

[E0816]

1.4_

0.8_

0.4_

_

_

_

SATRP1

*

*

*

*

*

Fig. 4. Genomic variability at the RPC2 locus after selective pressure. Southern analysis of KpnI-digested genomic

DNA (A) and (B) and PFG-separated chromosomes (C) from cell lines HZ400[E0816] and HZ0. The HZ400[E0816]

mutants were obtained after transfection of cosmid E08 into the heterozygous cell line HZ followed by culturing in

nourseothricin 400 mg.mlx1. HZ0 clones were isolated after culturing the mutants HZ400[E0816] without drug pressure

for 200 generations to eliminate the circular episome. The HZ400[E0816] mutants shown in lanes 4, 5 and 6 were cloned

from the cell line shown in lane 2. Likewise, HZ0 clones in lanes 1, 2, 3, 4, 6 and 7 originated from clone in lane 5.

The open circles indicate fragments bearing undisrupted copies of RPC2 ; the asterisks indicate the episomal

elements. The electrophoresis protocol that allowed the separation seen in A and B used a 0.5% agarose gel ; 45 V.cmx1

for 40 h. Mutant nomenclature is: HZ400[E0816] (RPC2/ ^rpc2::SAT400 [cLHYG16 E08^rpc2::SAT]) ; HZ0

(^rpc2::SAT0/ RPC2).

F. M. Squina and others 516

variation of KpnI fragments, hybridization of the

RP1 probe to EcoRV-digested DNA from these

heterozygous clones revealed a constant pattern for

both wild-type and disrupted loci (data not shown).

Hybridization signals of RP1 and SAT probes on

chromosomal blots of HZ0 clones were detected

exclusively in chromosome 20 (Fig. 4C), indicating

the absence of ectopic rearrangements of the targeted

locus. These results suggest that the appearance of

new KpnI fragments containing the undisrupted

RPC2 gene was not mediated by chromosomal

translocation or DNA amplification as previously

described for other loci (Genest et al. 2005).

Another striking feature revealed during the

attempts to generate a RPC2 chromosomal null

mutant was the rearrangement of the SAT gene into

the episomal molecule E08. As seen in Fig. 4A and B,

the clonesHZ400 [E0816], which were selected in high

doses of nourseothricin, bear an episome (indicated

as an asterisk) that is detected by both RP1 and SAT

probes. The integration of a marker originally pres-

ent in chromosome 20 of the heterozygous mutant

HZ into cosmid E08 was confirmed in episomal

molecules recovered from HZ400 [E0816] clones.

Fig. 5 shows the comparison between the cosmid E08

before transfection and after rescuing from HZ400

[E0816] clones. The only noticeable alteration in

their EcoRV restriction pattern is the increase in size

of the 3.3 kb fragment containing the wild-type

RPC2 locus. Southern blots hybridized with the RP1

and the SAT probes confirmed that the rescued

molecule carried the disrupted locus (Fig. 5). In or-

der to confirm that the rescued episome was not an

amplicon generated in HZ400 [E0816] clones, the

same blot was hybridized to a HYG probe, which

detected the cosmid backbone, the cLHYG vector

(Fig. 5).

As shown here and reported elsewhere for other

loci (Dumas et al. 1997), transfection of the intact

gene into a heterozygous clone, followed by the in-

crease in drug concentration, did not rescue null

mutants. Surprisingly, the disruption of chromoso-

mal copies of the RPC2 locus was selected in the

absence of high nourseothricin selective pressure

after transfection of cosmid E08 into heterozygous

clones. Southern analysis of KpnI-digested DNA

from representative clones suggested the disruption

of the RPC2 alleles (Fig. 6). In half of the selected

clones the RP1-positive KpnI fragments of chro-

mosomal origin also hybridized with the SAT probe

(clone LOH [E0816] ; Fig. 6). The other half of the

mutants (clone HZ [E0816] ; Fig. 6) carried at least 1

intact RPC2 locus and an untargeted cosmid E08.

The integration of the SATmarker into the episomal

molecule was also observed in mutant LOH [E0816].

As seen in Fig. 6, this clone bears an additional KpnI

fragment of y21 kb that is detected not only by the

RP1, but also by the SATprobe, which indicates that

some of the episomal molecules carry a disrupted

version of RPC2. Based on these results and on our

ability to dilute cosmid E08, Fig. 7 shows a schematic

representation of the possible configuration of RPC2

alleles and episomal molecules in clones selected

during the attempts to generate aRPC2 homozygous

mutant.

The heterozygous cell line and the mutants pres-

ented in Fig. 4A and Fig. 6 had a compromised

growth when compared to wild-type cells and en-

tered the stationary phase at a significantly (P<0.05)

RP1

1 2

SAT

1 2

HYG

1 21 2

-

-

--

-

-

-

--

-

-

-

--

-

6.5

4.3

2.3

23.2

2.0

Fig. 5. Targeting of the RPC2 locus within cosmid E08.

EcoRV restriction pattern and Southern analysis of

cosmid E08 before transfection into the parasite (lane 1)

and after rescuing from clone HZ400[E0816] (lane 2).

The probes used in Southern blot hybridization were

RP1, SAT and HYG, which detected the cosmid

backbone.

HZ

23.2_

9.4 _

~ 7 _ RP1 SATRP1 SAT

_

_

_

_

_

_

1 2 1 2

Fig. 6. Further disruption of RPC2 copies. Southern

analysis of KpnI-digested genomic DNA from the

heterozygous mutant HZ and cell lines HZ [E0816]

(lane 1) and LOH [E0816] (lane 2) selected exclusively in

hygromycin B after the transfection of cosmid E08 into

the HZ.2 mutant. Southern blots were probed with

fragments RP1 and SAT.

Shuttle mutagenesis and targeted disruption of Leishmania genes 517

lower density (Fig. 8A). This effect was not observed

in wild-type cells transfected with the cosmid E08,

which indicates that the phenotype was not caused

by the over-expression of the circular intact loci

(data not shown). Northern blots using total RNA

isolated from wild-type and mutant cell lines were

probed with RP1, SAT and actin gene (Fig. 8B).

Densitometry analysis of the data showed no sig-

nificant difference in the level of RPC2 transcript

between the heterozygous cell line and the wild-type

strain. However, mutant HZ400[E0816] had signifi-

cantly higher levels of both RPC2 and SAT tran-

scripts.

DISCUSSION

The availability of Leishmania mutants has greatly

contributed to extend our knowledge of this para-

site’s biology (Turco et al. 2001). In this work we

have used a shuttle mutagenesis approach to target a

telomere-located essential gene of L. major. The use

of the mariner in vitro transposition system led to the

generation of collections of reagents adequate for

targeted integration into the parasite’s genome. The

availability of a straightforward strategy for mutant

generation is particularly relevant if we take into

account that more than half of the predicted

Leishmania genes code for hypothetical proteins

(Ivens et al. 2005).

The integration efficiency into the L. major chro-

mosome 20 subtelomeric region was comparable to

that previously reported for L. major CC1 strain

(Cruz and Beverley, 1990; Cruz et al. 1991).

Although transcript levels of the targetedRPC2 gene

were not significantly reduced in the heterozygous

mutant, a low growth was observed, confirming that

first-round disruption mutants may be important

tools for gene dosage studies, as already demon-

strated for L. major and L. donovani Trypanothione

reductase (Dumas et al. 1997). Moreover, a cell line

bearing a selectable marker at the telomere can

be used in the study of the expression of telomere-

located Leishmania genes.

The generation of null mutants of Leishmania re-

quires successive and independent events of gene

inactivation. This can be achieved by rounds of

targeting or by one targeted disruption combined

with LOH, which uses high doses of antibiotics

to select a homozygous cell line and has the advantage

of saving selectable markers for targeted disruption

of other loci or for functional complementation

protocols (Gueiros-Filho and Beverley, 1996).

Instead of leading to a RPC2 null mutant, the use of

high doses of nourseothricin selected parasites with

multiple copies of the targeted locus, providing evi-

dence that this subunit of the Leishmania RNA

Polymerase III complex is essential for the parasite.

Although null mutants are readily generated for

HZ

X

E08

SAT

400 µµµµg.mL-1

LOH[E0816]

XXX

X

HZ[E0816]

XX

XX

HZ0

X

XX

HZ400[E0816]

SAT

400 µµµµg.mL-1HYG

16 µµµµg.mL-1

E08

No

drug

No

drug

Fig. 7. Schematic representation of the possible configuration of RPC2 mutants. Configuration of alleles and episomal

molecules in the heterozygous mutant HZ and mutant lines selected during the attempts to generate a RPC2 knockout.

The complete mutant nomenclature is: HZ (RPC2/RPC2/^rpc2::SAT20) ; HZ [E0816] (RPC2/^rpc2::SAT/^rpc2::SAT [cLHYG16 E08 RPC2].1) ; LOH [E0816] (^rpc2::SATLOH [cLHYG16 E08 RPC2/ ^rpc2::SAT]) and

HZ400[E0816] (RPC2/^rpc2::SAT400 [cLHYG16 E08 rpc2::SAT]).

F. M. Squina and others 518

non-essential genes in Leishmania, attempts to inac-

tivate vital genes may lead to extensive karyotype

alterations, which ensure the preservation of the

targeted gene (Cruz et al. 1993; Tovar et al. 1998;

Genest et al. 2005).

The supplementation with either an episome car-

rying the intact locus or the implicated metabolite

can be enough to guarantee the replacement of

chromosomal copies of an essential gene (Gueiros-

Filho and Beverley, 1996; Tovar et al. 1998).

Although RPC2 transcript levels were elevated in

cell lines complemented with the intact gene, the

generation of chromosomal null mutants of RPC2

was not possible even in the presence of high doses of

the selective drug. The genetic variability, which

granted the preservation of intact copies of RPC2 in

the selected mutants, did not involve chromosome

translocations or the generation of amplicons as

widely documented in Leishmania (Beverley, 1991;

Genest et al. 2005). The different KpnI fragments

containing the intact RPC2 locus may represent an

unexpected variation in the size of chromosome 20

subtelomeric region in these mutants. Size variation

between chromosome homologues has already been

described in Leishmania and may involve gene am-

plification and/or rearrangement of subtelomeric

repetitive sequences (Sunkin et al. 2000). Leishmania

telomeres contain varying amounts of typical sub-

telomeric repeats, such as LST-R (Myler et al. 1999;

El-Sayed et al. 2005) ; LCTAS (Fu and Barker, 1998)

and LST-R533 (Pedrosa et al. 2006). Therefore,

the variability observed in the mutant parasites

described in this work may have been mediated by

repetitive elements LCTAS and LST-R533, which

are present at the subtelomeric region of L. major

LT252 chromosome 20. Chromosomal ends of dif-

ferent protozoan parasites play relevant roles in

virulence and survival within their hosts (Borst

and Rudenko, 1994; Freitas-Junior et al. 2000; del

Portillo et al. 2001; Horn and Barry, 2005).

Subtelomeres of Trypanosoma brucei may contain

long tandem arrays of variant surface glycoprotein

(VSG) gene sequences and a single VSG expression

site, whose control is a key feature of antigenic vari-

ation in this parasite (Rudenko et al. 1998).

Chromosome size polymorphism in this trypanoso-

matid is mainly caused by a high degree of variability

in subtelomeres, which suggests that it can be par-

tially credited to the VSG system (Melville et al.

1999; Rudenko, 2000; Horn and Barry, 2005).

However, the subtelomeric regions of Leishmania

do not seem to be involved in such antigenic

variation mechanisms and are relatively shorter and

poorer in repetitive elements when compared to

0 20 40 60 80 100 120

106

107

Cel

l gro

wth

Hours

A

RP1 SAT ACT

_4.4_

B

HZ

HZ400

[E0816]WTHZ

HZ400

[E0816]WTHZ

HZ400

[E0816]WT

Fig. 8. Phenotypic analyses of RPC2 mutants. (A) Cell growth pattern of wild-type (closed square), the heterozygous

mutant HZ (open circle) and cell line LOH [E0816] (open triangle) cultivated in drug-free medium. (B) Northern blot

analysis of total RNA isolated from wild-type (WT), mutant HZ and cell line HZ400 [E0816] ; fragments RP1, SAT and

the actin gene were used as probes.

Shuttle mutagenesis and targeted disruption of Leishmania genes 519

other pathogenic trypanosomatids (El-Sayed et al.

2005).

The variability described here may provide new

information about the genome structure and control

of gene expression in Leishmania. The variation

forcing the maintenance of the loci in its original

chromosomal context suggests that tightly regulated

RPC2 expression is crucial for cell homeostasis in

L. major. In contrast to the endogenous locus, the

selected mutants indicated a marked preference for

the preservation of the SAT marker on a super-

numerary episome, as revealed by the unexpected

targeting of the supplementing circular molecule.

The SAT marker, originally present in the chro-

mosome of the heterozygous mutant, was found in

the cosmid E08 recovered from different trans-

fectants.DNArecombination between chromosomes

and circular episomes is commonly observed in

T. brucei (ten Asbroek et al. 1990) and is, in fact, a

distinguishing feature of the genetic tools available

for this trypanosomatid. On the other hand,

Leishmania episomal DNA is fairly stable and re-

arrangements between episomal elements are seldom

reported in this organism (Tobin et al. 1991).

Altogether, the mutants and events described in this

work may contribute to a better understanding not

only of the structure and function of the parasite’s

telomeres, but also of how their DNA recombination

machinery can be used in the control of gene ex-

pression.

As discussed above, the mutants generated in this

work confirm the genome plasticity of Leishmania

and provide new information about the parasite’s

genome maintenance. In addition, our data suggest

that the in vitro shuttle mutagenesis is a reliable

source of reagents for targeted disruption protocols

in this organism. The diversity of insertion events

that can be generated in a single reaction makes this

technologymore efficient than classical DNA cloning

methods (Beverley, 2003). Disruption fragments for

several genes can be easily produced, identified and

annotated into the genome. A pool of targeting

fragments can be introduced into the parasite by a

single mass-transfection experiment, allowing the

generation of several independent targeted mutants.

This is a systematic approach with inherent charac-

teristics of a large-scale post-genomic protocol, as

pointed out by Sakamoto et al. (2005). Therefore,

replacement events and rescued phenotypes require

further and more detailed studies. The mariner

toolkit developed for use in this parasite may gener-

ate an extensive collection of reagents, which include

not only functional knockout fragments but also

vehicles for targeted integration of fusion proteins

into the genome (Goyard et al. 2001; Robinson

et al. 2004; Augusto et al. 2004). Herein we have

shown that a simple method for the production of

reagents combined with the vast amount of sequence

information can greatly facilitate the generation of

engineered mutants. A comprehensive collection

of mutants will certainly help in the charac-

terization of the parasite’s virulence and in the

identification of potential targets for the control of

leishmaniases.

This work was supported by Fundacao de Amparo aPesquisa do Estado de Sao Paulo, FAPESP, 98/09805-0,99/12403-3; and UNDP/WORLD BANK/WHO SpecialProgramme for Research and Training in TropicalDiseases; FMS was sponsored by FAPESP; 01/02527-9.We thank Marlei Josieli Augusto for technical assistance.

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