Two casein kinase 1 isoforms are differentially expressed inTrypanosoma cruzi�

Carmenza Spadafora a, Yolanda Repetto b, Cristina Torres a, Laura Pino b,Carlos Robello a,1, Antonio Morello b, Francisco Gamarro a, Santiago Castanys a,*

a Instituto de Parasitologıa y Biomedicina ‘Lopez-Neyra’, Consejo Superior de Investigaciones Cientıficas (C.S.I.C.), Calle Ventanilla 11, 18001

Granada, Spainb Programa de Farmacologıa Molecular y Clınica, Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago de Chile,

Chile

Received 13 May 2002; received in revised form 23 July 2002; accepted 23 July 2002

Abstract

The cDNAs for two casein kinase 1 (CK1) homologues, TcCK1.1 and TcCK1.2 , have been isolated from Trypanosoma cruzi .

Both isoforms showed strong identity with other known CK1s. Their corresponding genes encode proteins of 312- and 330-amino

acid residues with apparent molecular weights of 16 and 37 kDa, respectively. TcCK1.1 is a two-copy gene while TcCK1.2 is

tandemly repeated, an arrangement not yet found in any other CK1. TcCK1.1 has been overexpressed in Escherichia coli and the

recombinant protein exhibited properties characteristic of the CK1 family. Northern blot indicated that both TcCK1s are expressed

differentially during the life stages of the parasite: the isoform TcCK1.1 shows low levels of mRNA expression in epimastigotes and

increased expression in trypomastigotes while TcCK1.2 presents an augmented expression in amastigotes as compared with the

other two life stages of the parasite. The CK1-like activity of amastigotes and trypomastigotes is significantly higher than that of

epimastigotes and, independent of the life stage of the parasite, a constitutive activity is observed which, in the epimastigote forms, is

found predominantly in the microsomal fraction. Also in the epimastigote forms, the CK1-like activity increases in the log phase of

growth of the parasites, and, through synchronization studies, this activity has been most conspicuously circumscribed to the S and

M phases of the cell cycle. # 2002 Elsevier Science B.V. All rights reserved.

Keywords: Casein kinase 1; Trypanosoma cruzi ; Cell cycle; Hymenialdisine; Differential expression

1. Introduction

The protozoan parasite Trypanosoma cruzi has a

complex life cycle, existing as extracellular flagellated

epimastigotes and metacyclic trypomastigotes in the

invertebrate host, and as trypomastigotes and intracel-

lular non-flagellated amastigotes in both phagocytic

(macrophages) and non-phagocytic cells such as fibro-

blasts, epithelial, endothelial muscle and nerve cells of

the vertebrate host. The intricate life cycle of T. cruzi

involves alternation between proliferative (epimastigotes

and amastigotes) and non-replicative (trypomastigotes)

stages. During its life cycle, the parasite encounters

changing environments that require rapid responses to

insure its survival. Little information is available about

the regulation of these complex cellular changes; how-

ever, as in higher eukaryotes, it is likely that these

protozoans control processes such as differentiation,

metabolism, growth, gene expression and other cellular

events through the reversible phosphorylation of pro-

teins.

Protein kinase CK1, also known as casein kinase 1

(CK1), is a family of multipotential Ser/Thr protein

kinases common to all eukaryotic cells. The ability to

Abbreviations: CK1, casein kinase 1; CKI-7, N -(-2-aminoethyl)-5-

chloroisoquinoline-8-sulphonamide; HD, hymenialdisine.�

Note: Nucleotide sequences data reported in this paper are

available in the GenBankTM database under the accession numbers

AF274060 and AF274059.

* Corresponding author. Tel.: �/34-958-805185; fax: �/34-958-

203911; http://www.ipb.csic.es

E-mail address: castanys@ipb.csic.cs (S. Castanys).1 Present address: Departamento de Bioquımica, Facultad de

Medicina, Universidad de la Republica, Montevideo, Uruguay.

Molecular & Biochemical Parasitology 124 (2002) 23�/36

www.parasitology-online.com

0166-6851/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 1 6 6 - 6 8 5 1 ( 0 2 ) 0 0 1 5 6 - 1

phosphorylate a wide variety of cellular proteins such as

cytoskeleton components, signaling molecules, meta-

bolic enzymes, proteins involved in mRNA translation,

enzymes involved in nucleic acid processing, and pro-teins involved in vesicular trafficking attests to the

importance of this multipotential protein kinase in cell

function [reviewed in 1 and 2]. CK1s are ubiquitous

proteins present in membrane, nucleus, cytoplasm,

vesicles, and cytoskeleton of eukaryotic cells. CK1s

have been generally isolated as monomer proteins with

variable molecular size (from 25 to 60 kDa) due to the

presence of many isoforms of the CK1 family encodedby different genes that differ in size. Seven members of

the CK1 family, and some of their splice variants, have

been cloned from mammalian cells (isoforms a, b, g1,

g2, g3, d, and o) [3�/6]. In Plasmodium falciparum a CK1

activity which is stage-specific regulated has been

described [7], and an ectokinase activity has been

associated with CK1 in Leishmania major [8] capable

of phosphorylating and inactivating molecules of thehuman complement system [9]. In Saccharomyces cer-

evisiae and Schizosaccharomyces pombe , eight different

CK1s have been characterized [10�/14]. One of these

genes, HRR25 of S. cerevisiae , has been described as a

CK1 involved in DNA repair and cell cycle control. In

S. pombe , the CK1 homologues Hhp1 and Hhp2 play

similar roles since single and double mutations in the

Hhp genes reveal DNA repair defects in these cells.Interestingly, RAG8, the CK1 isoform of Kluveromyces

lactis , seems to be an essential gene based on the

lethality of the rag8 null mutation [15]. Most recently,

Calabokis et al. [16] reported the biochemical character-

ization of a partially purified CK1-like activity in T.

cruzi , but the CK1 genes of this trypanosomatid had not

yet been isolated and a study of its function had not

been initiated until now. Due to the high degree ofconservation of this enzyme between lower and higher

eukaryotic isoforms, it is likely that findings on CK1

activity and function will hold for all members of this

subfamily of kinases and that the study of lower

eukaryotes will help to shed some light on the increas-

ingly interesting role of CK1 in mammals, particularly

with respect to its participation in neuropathologies [17]

circadian rhythm [18], and developmental processesthrough a positive regulation of the Wnt pathway [19].

The differences found, nonetheless, represent the key

point in which an important therapeutic window may

potentially open for the treatment of this parasitic

disease.

Here we report the molecular characterization, and

stage specific expression of T. cruzi casein kinase

homolog 1 (TcCK1.1 ) and homolog 2 (TcCK1.2 ) anda partial biochemical analysis of TcCK1.1. We under-

took the study of the relationship of CK1 to the

proliferative activities of T. cruzi . Also, the inhibitory

effectiveness of the marine sponge-isolated inhibitor of

CK1, hymenialdisine [20], was tested on T. cruzi

parasite extracts as well as on recombinant TcCK1.1.

2. Materials and methods

2.1. Chemicals

[g-32P]ATP (5000 Ci mmol�1) was obtained fromAmersham International; Ni2�-nitrilotriacetic acid

agarose (Ni-NTA) column was purchased from Qiagen;

mouse 6X-His monoclonal antibody was from Clontech;

pET22b�/ His-tagged expression vector was from No-

vagen; N -(2-aminoethyl)-5-chloroisoquinoline-8-sul-

phonamide (CKI-7) was from Seikagaku America;

phosphocellulose P81 was from Whatman; Propidium

iodide (PI) and the Casein Kinase-1 peptide substratewere from Sigma. All other chemicals were of analytical

reagent grade.

2.2. Parasite culture, subcellullar fractionation, and

synchronization

The Y strain of T. cruzi was used throughout most of

our work, but in some cases CL-Brener was also used.

Epimastigotes were grown in liver infusion tryptose

(LIT) medium supplemented with 10% heat-inactivated

fetal calf serum (FCS) at 28 8C. Trypomastigotes wereobtained according to a method described by Zulantay

and co-workers [21]. Amastigotes were obtained by

incubating recently released trypomastigotes in LIT

medium under 5% CO2 for 24 h at 37 8C [22]. For

subcellular fractionation, epimastigotes were grown to

logarithmic phase and harvested by centrifugation

(110�/g for 5 min) and washed twice in PBS. The

parasites were then resuspended in Buffer L. (50 mMHepes, pH 7.8, 1 mM PMSF, 0.7 mg ml�1 pepstatin A,

and 5 mg ml�1 each of leupeptin, aprotinin and

antipain) and disrupted by two sonications of 30 s on

a Vibra Cell Sonifier, with a 30 s interval between each

sonication, using the micro (20s) probe on the 50% duty

pulse setting; the sonicated extract was centrifuged at

16 300�/g for 15 min at 4 8C. The pellet, called P-10

was stored at �/20 8C until use and the supernatant wascentrifuged further at 105 000�/g for 1 h. This step left

us with a microsomal pellet fraction and a cytosolic

supernatant. Synchronization of the cell cycle in epi-

mastigotes, using hydroxyurea, was performed as de-

scribed [23]. Briefly, cells from the 6th day of culture

were incubated for 24 h in fresh LIT medium containing

10% FCS plus 0.02 M hydroxyurea. Following this

treatment, cells were collected, washed twice with PBS,and resuspended in fresh medium containing 10% FCS.

Aliquots of about 6�/107 parasites were then collected

at different times and their cell cycles analyzed by flow

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/3624

cytometry to confirm synchronization of the parasites as

described [24] using PI to analyze the DNA content.

2.3. Library screening, subcloning, and sequence analysis

A T. cruzi (Y strain) lZAP† (Stratagene, La Jolla,

CA) cDNA library was used. The CK1 conserved probe

was obtained by PCR using degenerate primers from the

CK1 conserved sequences in motif 1 (See Fig. 1)

[sense: CT(G,C)CT(G,C)GG(C,G)CC(G,C)TC(G,C)

CT(G,C)GA] and motif 3 [anti sense: TT(G,C)

AG(C,G)CCCTGCCA(G,C)GG(G,C) AG] on the basis

of sequence alignment of 11 CK1 isoforms from eight

species [25]. The PCR was run by cycling 10 s at 94 8C,

30 s at 58 8C, and 60 s at 72 8C, over 30 cycles, using

genomic DNA from the Y strain as the template. This

PCR yielded a product of 603 bp, which was sequenced

on both strands by the dideoxy chain-termination

Fig. 1. Sequence alignment of CK1 isoforms from different species. TcCK1.1 (T. cruzi ), TcCK1.2 (T. cruzi ), HuCK1d (Human d-isoform), HuCK1e

(Human o-isoform), Hhp1 (S. pombe ), PfCK1 (P. falciparum ), DmCK1 (D. melanogaster ), AtCK1 (A. thaliana ). Protein sequences were aligned for

maximum identity by introducing gaps (represented by dots) using the University of Wisconsin GCG PILEUP program. Identical residues in all

CK1s described to date are shaded in gray, and identical residues in the represented species which are conserved in the rest of the CK1 family

members are shaded in black. The signature sequences (motifs) specific to the CK1 family [1] are shown with a double line on top and numbered (1, 2,

3). The primers designed to obtain the CK1 probe were taken from motifs 1 and 3. The three residues exclusive to T. cruzi CK1s are boxed. The

single and dotted lines show the kinesin homology domain and the nuclear localization signal, respectively.

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/36 25

method in a 373 Automated DNA sequencer (Applied

Biosystems, USA) and used as a probe to screen the T.

cruzi cDNA library.

Approximately 100 000 phage plaques were screened

with this probe using 50% formamide at 42 8C accord-

ing to standard procedure [26]. Filters were washed

twice in 2�/SSC (1�/SSC is 0.15 M NaCl, 0.015 M

sodium citrate), 0.1% SDS at 42 8C; the positive

plaques were purified and their DNA isolated by in

vivo excision with the ExAssistTM Helper Phage (Stra-

tagene), which generated subclones in the pBluescript†

SK-phagemid (Stratagene) in the Escherichia coli strain

XOLR. The subclones were sequenced by using uni-

versal and overlapping oligonucleotide primers and

analyzed by using the BLAST algorithm [27] and the

University of Wisconsin GCG software package [28].

2.4. Parasite genomic organization and mRNA

expression

T. cruzi DNA was obtained by phenol extraction [29].

It was subjected to restriction analysis, Southern blot,

and hybridization through classical methods [26]. The

blotted DNA was hybridized to the 603 bp CK1 probe

and to the specific TcCK1.1 and TcCK1.2 probes,

labeled by random priming. Total RNA from T. cruzi

amastigote, trypomastigote, and epimastigote forms was

obtained by Trizol (Invitrogen�/Life Technologies) ex-

traction. It was size-separated on formaldehyde-agarose

gels, blotted, and hybridized to the same specific probes

described above, and to the TcCK1 probe that com-

prises the complete TcCK1.2 gene, 88% identical to

TcCK1.1 . Hybridization and washings were conducted

at 42 8C as described [26]. Hybridizations with T. cruzi

b-tubulin and 18S rDNA probes, and ethidium bromide

images were employed for normalization of the North-

ern blots. The radioactive signals were quantified using

InstantImager electronic autoradiographies (Packard,

Meriden, CT), and the ethidium bromide stainings

were captured with a Gel Printer Plus Station (Scion

Image, Scion Corp. Maryland, USA) and analyzed with

the 1-D Manager (T.D.I., S.A., Madrid, Spain) soft-

ware. Low-melting point agarose blocks of T. cruzi

epimastigote forms were prepared as described [30].

Total chromosomal DNA was separated by a contour-

clamped homogeneous electric field (CHEF) in a LKB

2015 Pulsaphor System apparatus (Pharmacia LKB,

Sweden), using 1% agarose gels and 0.5�/TBE buffer

(1�/TBE is 45 mM Tris, 45 mM boric acid, 1 mM

EDTA, pH 8.3) at 13 8C. The running conditions were

250-, 500-, 750-, and 1000-s pulse times for 24 h at 90 V.

DNA standards were from the S. cerevisiae strain S13

(Roche, Germany). Transferred DNA was hybridized to

the TcCK.1.1 and TcCK1.2 probes.

2.5. Expression and purification of recombinant TcCK1.1

The TcCK1.1 open-reading frame from the cDNA

clone was amplified by PCR, employing the ExpandTM

Long Template PCR System kit (Roche) by cycling 10 s

at 94 8C, 30 s at 60 8C, and 60 s at 68 8C over 30

cycles, using the specific primers 5?-ctggatccgAT-

GAACCTAATGATTGCAAACAG-3? and 5?-acaagct-

tATTGTTAGACAATTCTTTCTCTTC-3? (lowercase

letters represent non-coding sequences containing the

restriction sites BamHI and HindIII, respectively, in-

troduced to facilitate cloning of PCR products). ThePCR product was digested with BamHI and HindIII

and ligated to the BamHI/HindIII sites of E. coli vector

pET22b�/ for protein expression. Double-stranded

DNA sequencing was performed to confirm the correct

sequence after PCR amplification and ligation. The

construction was called TcCK1.1/pET22b�/. E. coli

strain C41(DE3), derived from E. coli BL21(DE3),

(generously provided by Dr J.E. Walker, Laboratoryof Molecular Biology, Hills Road, Cambridge, UK) was

used to express the TcCK1.1 protein. Transformed cells

were incubated for nearly 4 h at 37 8C in LB medium

and induced for periods of either 4 or 18 h with 0.5 mM

isopropyl b-thiogalactoside at the respective tempera-

tures of 37 or 19 8C, then harvested by centrifugation,

washed twice with PBS, and stored at �/80 8C until use.

Aliquots of the E. coli extracts were loaded into a 12%SDS-PAGE according to Laemmli [31] for analysis.

Frozen pellets of induced bacterial cells were resus-

pended in 5 ml buffer L and 10 mM MgCl2 per gram of

wet weight. Lysozyme was added to a final concentra-

tion of 1 mg ml�1 and incubated for 30 min at room

temperature. The cell suspension was sonicated five

times for 30 s with 30 s intervals and incubated with

DNase I (200 mg ml�1) for 1 h at 4 8C, then centrifugedat 30 000�/g for 20 min at 4 8C to yield a clear

supernatant that was loaded directly onto a 2 ml Ni-

NTA agarose column. The column was previously

equilibrated in ten volumes of 50 nM Hepes, pH 7.8,

150 mM NaCl, 1% Triton X-100, and 10% glycerol.

After protein binding, the column was loaded with 50

bed volumes of wash buffer (50 mM Hepes, pH 7.8, 0.7

M NaCl, 10% glycerol, 40 mM imidazole, 2 mM b-mercaptoethanol and 0.05% Triton X-100) The protein

was eluted with 50 mM Hepes, pH 7.8, 150 mM NaCl,

and 10% glycerol with an increasing linear gradient of

imidazole (from 40 to 200 mM). 500 ml protein fractions

were collected and their aliquots analyzed on 12% SDS-

polyacrylamide gels. The fractions were stored at �/

2 8C; under these conditions, the enzyme retained

activity for at least 1 year. The SDS-polyacrylamidegels containing the fractions aliquots were electro-

transferred onto Millipore PVDF membranes. Blots

were incubated with the mouse anti-hexahistidine mono-

clonal antibody at a 1:2500 dilution in Buffer A [TBS

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/3626

(25 mM Tris, 2.7 mM KC1, and 137 mM NaCl at pH

7.4) 1% BSA, 0.1% Tween 20], for 1 h at room

temperature. After washing with TBS, bound antibodies

were detected by incubating the blot for 30 min with theHRP-conjugated anti-mouse IgG (Sigma) at a 1:3750

dilution in Buffer A, and developed with the enhanced

chemoluminiscence (ECL) kit from Amersham pharma-

cia biotech. Similar experiments to clone and express

TcCK1.2 were unsuccessful, yielding no product of the

expected size.

2.6. Protein kinase assays

The assay mixture (30 ml) contained: 50 mM Hepes,

pH 7.8, 150 mM NaCl, 7 mM MgCl2, 0.5 mM DTT,

[g-32P]ATP (2�/100 mM, specific activity 500�/1000 cpm

pmol�1), 1�/16 mg ml�1 dephosphorylated b-casein

100�/800 mM Casein Kinase-1 peptide substrate

(RRKDLHDDEEDEAMSITA), and, in most cases,

two units of recombinant TcCK1.1 from the purified

fractions eluted from the Ni-NTA agarose column. Aunit of enzyme activity (U) is defined as the amount of

enzyme that catalyzes the incorporation of 1 pmol of

phosphate into the substrate per minute. When testing

the activity of the recombinant protein, a bacterial

control was included to counteract the effect of en-

dogenous kinases. For determining inhibition of CK1

activity, heparin or CKI-7 [32] (a specific CK1 inhibi-

tor), were also used in the mixture as follows: heparinwas added in concentrations ranging from 0.15 to 30 mg

ml�1, and to assay the effect of CKI-7 we used it at 100

mM. We have also tested a different CK1 inhibitor,

hymenialdisine (HD), a marine sponge-derived natural

product (generously provided by Dr L. Meijer and Dr

G.R. Pettit from the Station Biologique, CNRS, Rosc-

off, France, and The Cancer Research Institute, Arizona

State University, AZ, USA, respectively) [20] in con-centrations ranging from 5 to 100 nM. In the experi-

ments where total parasite extracts were employed, the

parasites were collected and washed three times with

PBS and then resuspended in buffer L containing 0.1%

Triton X-100. The amount of protein used per sample in

these experiments was approximately 10 mg. In all cases,

reactions were initiated by the addition of the

[g-32P]ATP, except for the Km assays for ATP, wherethey were initiated by the addition of the kinase.

Incubations were carried out for 10 min at 30 8C and

the reaction was terminated by adsorbing the solution

onto 2.0 cm2 of Watman P81 phosphocellulose filters as

described [6]. The filters were washed extensively in 75

mM phosphoric acid and dried. The radioactivity

retained in the filters was quantified by duplicates in a

b-scintillation counter. Control values for correctingbackground and endogenous phosphorylation were

obtained by reactions run in the absence of either the

enzyme or substrate in the assay mixture. These values

were subtracted from the sample activity. In addition to

the use of double reciprocal plots (Lineweaver�/Burk

plots), the kinetic constants of all substrates were

determined by non-linear regression data fit analysis ofthe Michaelis�/Menten curve, as calculated by the

GraphIt software with a robust weighting of the data.

3. Results

3.1. Cloning of TcCK1.1 and TcCK1.2 gene

Since proteins belonging to the CK1 family containseveral hallmark amino acid sequence identifiers that

distinguish these proteins from other kinases [33], we

used these conserved sequences to design primers to run

a PCR amplification of the conserved part of the T.

cruzi CK1 gene(s). This PCR product of 603 bp was

sequence-analyzed and confirmed to correspond to a

CK1 showing a 79% homology with human CK1d. The

radiolabeled fragment was subsequently employed as aprobe to screen a cDNA library of the Y strain of T.

cruzi. Eight positive clones were isolated and purified.

The cDNA clones were sequenced and confirmed to

contain two isoforms of the T. cruzi CK1 genes. One of

them contained the TcCK1 homolog 1 (TcCK1.1 ), 936

nucleotides long and coding for a 312 amino acid

polypeptide of approximately 36 kDa, and the others

contained the TcCK1 homolog 2 (TcCK1.2 ), 990nucleotides long and coding for a 330 amino acid

polypeptide of approximately 37 kDa. The isoforms

are 88% identical to each other (94% in their catalytic

domains) and differ mainly in their carboxyl-terminal.

Homolog 2 has a 35-amino acid extension beyond the

kinase domain, whereas homolog 1 has a shorter 19-

amino-acid tail (Fig. 1). TcCK1.1 and TcCK1.2 are

about 65% identical to human CK1d and CK1o, and toS. pombe Hhpl. They are also highly homologous to

CK1s of other species, like Arabidopsis thaliana (63%),

Drosophila melanogaster (59%), and P. falciparum

(58%). With respect to the amino acids of interest for

the tertiary structure, the three signature sequences

specific to the CK1 family are present in TcCK1.1 and

TcCK1.2 (See also Fig. 1). The signature sequence of

loop L-EF (LPWQGLKA) (residues 214�/221, takingTcCK1.1 as its reference) is conserved in all organisms

described up to this point, with the exception of

TcCK1.1, described here, in which the lysine is replaced

by proline. (For a description of the nomenclature used

for CK1 proteins see Xu et al. [34]). The nuclear

localization sequence described by Gross and Anderson

[1], TKRQKY (on human CK1d), corresponds to the

sequence TKQEKY (residues 223�/228) in both TcCK1homologues. There is also a kinesin homology domain

[1], HIPYR (again, in human CK1d), which is conserved

in both T. cruzi homologues (residues 167�/171). On the

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/36 27

other hand, there is an insertion of three residues, GGV,

in the nucleotide binding domain of T. cruzi (residues

63�/65), absent in other CK1s published (Fig. 1).

3.2. Genomic organization and chromosomal localization

of TcCK1 genes

Preliminary digestion with restriction enzymes of

genomic DNA from the Y strain of T. cruzi and

subsequent hybridization with the 603 bp CK1 con-served probe suggested the existence of different CK1

genes, one of which seemed to be a multicopy gene (Fig.

2A). When the two isolated genes had been cloned and

sequenced, we designed specific probes for each one of

them. For TcCK1.1 , we designed a probe based on

nucleotides �/166 to 37; and for TcCK1.2 we used a

specific probe based on nucleotides 909 to 1260. These

labelled fragments were used to hybridize a Southern

blot of T. cruzi genomic DNA digested with different

endonucleases. Each hybridized Southern blot revealed

different restriction patterns as shown in Fig. 2 (B, C).

In the case of TcCK1.2 , except for one lane, the entire

DNA hybridized to bands of the same size when it was

digested with different enzymes that cut the gene once,

except for KspI (Fig. 2C). In the case of digestion with

KspI a fragment of approximately 600 bp was obtained

due to an extra restriction site in the gene (Fig. 2C, lane

1). The hybridization pattern suggests that TcCK1.2 is

indeed genomically repeated in tandem, which is not the

case of TcCK1.1 , a seemingly two-copy gene per haploid

genome (Fig. 2B). To further confirm that TcCK1.2 is a

multicopy gene, we made a partial digestion of genomic

Fig. 2. Genomic organization of TcCK1 genes from T. cruzi . 5 mg per lane of genomic DNA from the Y strain were digested with different

restriction enzymes, electrophoresed, blotted and hybridized with different probes. (A) a probe corresponding to a 603-bp CK1 coding region (see

Experimental Procedures), lane 1, Ksp I; lane 2, Sac I; lane 3, Kpn I; lane 4, Sca I. (B) TcCK1.1 specific probe. Lane 1, Xho I; lane 2, Sca I; lane 3, Ksp I;

lane 4, Pvu II. (C) TcCK1.2 specific probe. Lane 1, Ksp I; lane 2, Sac I: lane 3, Kpn I; lane 4, Sca I. (D) 5 mg/lane of genomic DNA were digested with

Kpn I at 2, 4, 8, 15, 30, 60, and 90 min (lanes 1 to 7), and hybridized with the TcCK1.2 specific probe, showing the typical ladder pattern of a

tandemly-repeated gene. None of the enzymes used cut the probes. Size markers (Kb) are derived from l phage DNA digested with the restriction

endonucleases Hind III and fX174 digested with Hae III. (E,F) Chromosomal localization of TcCK1.1 and TcCK1.2 genes: Chromosomes of

epimastigote forms were separated by Contour-clamped homogeneous electric field (CHEF) under conditions described in Experimental Procedures.

The gel was blotted and hybridized with the specific probes TcCK1.1 (E) and TcCK1.2 (F). Size markers (Mb) were Yeast Chromosomes PFG

Marker from Biolabs. (O) represents the well location.

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/3628

T. cruzi DNA with KpnI. The samples were run on

agarose gel, blotted, and hybridized with the TcCK1.2

specific probe. This partial digestion shows the multi-

plicity of the tandem-repeated isoform, with a minimumof 14 or 15 copies (Fig. 2D). To determine the

chromosomal localization of CK1 genes, T. cruzi

chromosomes were separated by pulse-field gel electro-

phoresis and hybridized with the specific probes de-

scribed above. The results indicate that, in the Y strain,

both isoforms, TcCK1.1 and TcCK1.2 , are each located

on two chromosomes larger than 2 Mb (Fig. 2E, F).

3.3. Stage-specific mRNA expression and activity

We investigated the expression of both genes during

the different life stages of the parasite. Total RNAs from

amastigote, trypomastigote, and epimastigote forms of

the CL-Brener strain of T. cruzi were extracted and size-

separated on formaldehyde-agarose gels. They were

blotted and hybridized to the TcCK1.1 and TcCK1.2

specific probes, and to the TcCK1 probe. Transcripts ofabout 1300 bp, the size predicted from the cDNA clones,

appeared in both filters, but the stage-specific expression

was different for each. In the case of TcCK1.1 (Fig. 3A),

there was low expression in the epimastigote stage,

compared with a more than 30-fold increase (Fig. 3D) in

the trypomastigote stage (the blot was overexposed in

order to detect the transcript in the epimastigote lane)

(Fig. 3A). For TcCK1.2 (Fig. 3B), the epimastigote andtrypomastigote forms show a similar expression, while

the amastigote stage reveals a 2-fold increase in expres-

sion (Fig. 3D). We hybridized the Northern blots with

the TcCK1 probe that recognizes both TcCK1.1 and

TcCK1.2 isoforms, and found that in the amastigote

and trypomastigote forms, the mRNA of TcCK1s are

expressed more than three times in the first form and

almost two times in the second, as compared with theepimastigote forms (Fig. 3C).

The CK1-like activity of the parasite extracts was

highest in amastigotes, with minor differences in trypo-

mastigotes, while the activity in epimastigotes ranged

from 57 to 68% of that of amastigotes, depending on the

state of replication of the culture from which the

samples were taken (see below) (For this comparison,

only CK1 activities prior to the stationary phase ofgrowth were taken into account) (Fig. 3E).

3.4. Parasite subcellular fractionation

Epimastigote forms of the Y strain of T. cruzi were

lysed and separated into nuclear, cytosolic and micro-

somal fractions by differential centrifugation. Analysis

was made of the CK1-like activity of these fractions,revealing that the most significant part of the activity

was localized within the microsomal fraction (Table 1).

More than 53% of the activity was retained in the

microsomal pellet while the supernatant of the

105 000�/g centrifugation had 16% of the total activity,

and the P-10 (nuclear and mitochondrial pellet) ac-

counted for only 7.5% of the total activity.

3.5. CK1-like activity during T. cruzi growth

We analyzed the CKl-like activity during the cell

growth of epimastigote forms of T. cruzi on Y and CL-

Brener strains. As shown in Fig. 4, activity was observed

on the initial day of growth. This activity continues to

increase until parasites reach the late log phase, at which

point a drop in activity is observed. The analysis of theCK1-like activity in two strains with very different

growth rates, with CL-Brener being slower than Y,

reveals that the highest doubling time of the parasite

coincides with a higher specific CK1-like activity (See

Fig. 4).

3.6. TcCK1 expression and activity along cell cycle

progression

T. cruzi epimastigotes were synchronized and mon-

itored by flow cytometry analysis (not shown). The

mRNAs from both TcCK1.1 and TcCK1.2 were ana-

lyzed by quantification of the radioactive signals nor-

malized against the signal of b-tubulin and 18S rRNA

(Fig. 5A). The mRNA expression of TcCK1.1 follows a

pattern similar to that of the CKl-like activity of theparasite extracts, while mRNA from TcCK1.2 increases

after release from hydroxyurea arrest and maintains a

similar expression throughout the cell cycle (Fig. 5B).

After release from the blockade (t�/0) (Fig. 5C), the

epimastigote forms present a gradually increasing CK1-

like activity as the parasites enter the S phase, until t�/

10 h when DNA replication reaches a maximum. By t�/

12 h most of the DNA synthesis is completed; concur-rently, the CK1-like activity decreases until t�/15 h

when most of the cells are in early G2/M. A sharp

increase in enzyme activity is observed at t�/18 h (late

G2/M), when epimastigotes are in or are leaving mitosis

according to flow cytometry analysis (not shown).

3.7. Expression and purification of recombinant TcCK1.1

TcCK1.1 was cloned in the pET22b�/ vector and

expressed in E. coli C41(DE3). Induced extracts dis-

played a moderate production of an approximately 36

kDa protein (Fig. 6A) which remained in the pellet

fraction of the crude lysate (Fig. 6B). The size of the

overexpressed protein complied with the predicted

molecular weight of 36 kDa for the TcCK1.1 open-

reading frame polypeptide. Probably due to the effect ofCK1 on bacterial functions, low amounts of the CKl

recombinant protein are obtained, making protein

concentrations difficult to measure, as previously re-

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/36 29

ported; nonetheless, it has a very high specific activity,

as reported by Klimczak and co-workers [35]. To

optimize the yield of the TcCK1.1 recombinant protein,

we reduced the temperature for induction from 37 to

19 8C and increased incubation time to 18 h. Immuno-

detection of the soluble fraction failed to produce a

signal. We proceeded with the purification of this

soluble fraction by incubating it in a Ni-NTA agarose

column and eluting with a linear gradient of imidazole.

The activity of the fractions was determined by their

kinetic functionality, as described below; those with the

highest activity were pooled, concentrated, and analyzed

by SDS-PAGE and subsequent Coomasie staining (Fig.

6B), Western blotting, and immunodetection. Using this

Fig. 3. Stage-specific expression of TcCK1 mRNA. Total RNA (20 mg) front mid-to-late log epimastigote (E), amastigote (A), and trypomastigote

(T) forms of the life cycle of the CL-Brener strain of T. cruzi were hybridized with the TcCK1.1 (A), TcCK1.2 (B), and the TcCK1 specific probes

(C). The ethidium bromide stainings of the rRNA s are shown in the lower panels. Standard RNA markers (kb) were from Promega. (D) Fold CK1

mRNA expression with respect to the epimastigote forms of the parasite, according to normalization with densitometry analysis of rRNAs (See

Experimental Procedures). (E) Different forms of the parasite (CL-Brener strain) were obtained as described in Section 2. Supernatant samples of the

13 000�/g centrifugation were analyzed in duplicate with respect to their specific CK1 activity. The data is the result of a representative experiment.

For the epimastigotes, the number calculated was the media of the activity during 6 days of the logarithmic phase of growth.

Table 1

CKl-like activity of different cellular fractions of T. cruzi

Fraction Specific Activity

(pmol min�1 per mg)

Total Activity

(pmol min�1)

Sonicated total extract 3180 24 887

P-10 2460 1877

Cytosolic supernatant 1406 4007

Microsomal pellet 3755 13 261

Epimastigotes from the T. cruzi Y strain were fractionated as

described in Experimental Procedures. CK1-like activity was measured

in the different fractions. The kinetic assays were performed by

duplicate with standard deviation (S.D.) below 10%.

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/3630

strategy, we detected the presence of the recombinant

protein (Fig. 6C).

3.8. Enzymatic activity of recombinant TcCK1.1

Recombinant TcCK1.1 was used for enzymatic assays

according to the method previously described. The

fractions eluted from the Ni-NTA agarose column

were analyzed on the basis of their CK1 activities.

Those fractions within the peak of activity were pooled,concentrated, and used for the kinetic assays. We found

that the apparent Km for b-casein and the specific

synthetic peptide Casein Kinase-1 substrate were 5.7 mg

ml�1 and 128 mM, respectively. For ATP, the TcCK1.1

protein exhibited an apparent Km of 56 mM. When the

specific synthetic peptide Casein Kinase-1 was used as

substrate, an inhibition of nearly 40% was obtained at

100 mM CKI-7 with 100 mM ATP as well. Heparin,being a specific inhibitor of CK2 proteins, has been used

as a means of distinguishing between CK1 and CK2

protein kinases [25]. We observed that heparin slightly

diminished TcCK1.1 activity, by about 10% at the

highest concentration used (30 mg ml�1). The other

compound tested, HD, is a natural and potent inhibitor

of CK1 [20]. When the effect of HD on CK1 was

studied, we observed that CK1 activity was significantly

inhibited when using parasite extracts (IC50, 23 nM), byas much as when recombinant TcCKl.l (IC50, 13 nM) is

used (data not shown). Thus, in terms of general

biochemical parameters, recombinant TcCK1.1 exhib-

ited properties characteristic of the CK1 family of

enzymes.

4. Discussion

We have isolated two genes in T. cruzi that encode

proteins homologous to the CK1 family. Both TcCK1

genes seem to be localized in two chromosomes,however, bearing in mind the diploidy of this organism,

and since allele polymorphisms have been described in

this trypanosomatid [36], they are probably in two

Fig. 4. CK1-like activity throughout cell growth of epimastigote forms of T. cruzi . Using epimastigote forms of (A) Y and (B) CL-Brener strains of

T. cruzi , we analyzed CK1 activities throughout the cell growth of parasites using the Casein kinase-1 specific substrate. Circles represent the number

of parasites per ml and squares represent CK1-like specific activity.

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/36 31

Fig. 5. Expression and CK1-like activity along the cell cycle of T. cruzi . (A) Expression of TcCK1.1 and TcCK1.2 mRNAs. Aliquots of

synchronized T. cruzi Y epimastigotes were taken at times 0, 6, 8, 12, 16, 18, and 24 h. Time zero represents the moment in which the parasites ant

taken out of the hydroxyurea medium (see Experimental Procedures). Their RNAs were extracted, approximately 25 mg of each were transferred to a

nylon filter and hybridized with the specific isoform probes TcCK1.1 and TcCK1.2 to determine the variations of expression throughout the cell

cycle. The blot was normalized using the b-tubulin and the 18S rRNA probes. (B) Relationship of the mRNA expression and activity of the TcCK1

isoforms and the cell cycle. From the two upper panels, each time point represents the fold change in mRNAs levels (relative to the 0 h time point)

with respect to their normalization with either b-tubulin (solid lines) and 18S rRNA (dotted lines) specific probes. The lower panel shows CK1-like

activity in synchronized parasites. For enzymatic activity, samples of the parasites were taken out at times. 0, 2, 4, 6, 8. 10, 12, 15, 18, 21, and 23 h,

and their CK1-like activity determined using the Casein kinase-1 peptide substrate (See Experimental Procedures). Experiments were repeated three

times and gave essentially the same profiles as the experiment shown here.

Fig. 6. Overexpression and purification of recombinant TcCK1.1, (A) Stained Coomasie Blue SDS-PAGE gel of total bacterial proteins from E. coli

cells transformed with pET22b�/ (lane 1) and TcCK1.1/pET22b-(lane 2) after IPTG induction. (B) Stained gel containing supernatant (lane 3) and

pellet (lane 4) fractions from the total crude extracts of TcCK1.1/pET22b�/; purified 6XHis-tagged proteins from Ni-NTA agarose column: a

fraction with the maximal CK1 activity (lane 5) and a fraction without CK1 activity (lane 6). (C) Western blot analysis of total induced extracts (lane

7), a fraction with the maximal CK1 activity (lane 8) and a fraction without CK1 activity (lane 9) using a monoclonal anti 6X-His antibody (at 1:1000

dilution) as described in ‘Experimental Procedures’. Arrows indicate the position of TcCK1.1. Molecular mass standards (kDa) were from Bio-Rad.

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/3632

alleles of the same chromosome, with different sizes. An

important characteristic of the TcCK1 isoforms is that

even though one of the genes, TcCK1.1 , is a two-copy

gene (per haploid genome), the other, TcCK1.2 , is amulticopy gene, the only one described to date.

CK1 is a family of Ser/Thr kinases which are highly

conserved in their catalytic domain; they are identical by

50% or more, presenting the most differences in their

NH2 and COOH termini. It is these sequence differences

upon which most isoform classification is based. The

CK1 isoforms have been clustered in three main

branches [1]: enzymes found exclusively in the cyto-plasm, where they interact with the plasma membrane,

and which are represented by YCKs and Cki1; enzymes

found largely in the nucleus, regulating DNA repair and

best exemplified by HRR25; and finally, enzymes which

appear to be widely distributed throughout the cell,

performing such diverse functions as regulation of

mitosis or signal transduction, and represented by

CK1b and CK1a. The TcCK1 homologues are similarto many of the known CK1 isoforms from mammals

and yeast, and cannot be ascribed a function based on

sequence similarity alone. Both TcCK1 genes contain

almost all of the residues that are characteristic of these

protein kinases, but there are some differences. The

most remarkable aspect of the sequence of the TcCK1

isoforms is a unique three-residue insertion within the

catalytic domain, not present in the rest of the familydescribed. This insertion would occupy a place in the

loop joining the aA helix with the b4 strand (L-A4) [34].

The loop is localized behind the ATP binding site, where

the three residues could influence nucleotide binding.

This sequence insertion could explain some relative

biochemical differences found in TcCK1.1. Since the

triphosphate subsite is highly conserved throughout the

kinase superfamily, the differences found in the residuesthat line the purine pocket are key to the design of

specific agents that could interfere with the enzyme

activity. In the search for successful drugs against

parasitic diseases, the enzyme targeted should be suffi-

ciently different from the host protein so that the drug

used only affects the parasite. Significantly, Knockaert

and colleagues [37] described how a matrix of purvala-

nol, a purine derivative, bound some protozoan CK1s,T. cruzi among them, and not those CK1s from other

species. Could these three residues lie behind such an

observation in the case of this trypanosomatid?

While looking for specific inhibitors of cyclin-depen-

dent kinases (CDKs), Meijer and other colleagues

reported the finding of hymenyaldisine (HD) [20], an

inhibitor of human CDK1, CDK5, GSK3-b and CK1,

with IC50 values of 22, 28, 10, and 35 nM, respectively.We have tried HD on T. cruzi CK1, obtaining an IC50

of 23 nM for the parasite CK1-like activity and 13 nM

for recombinant TcCK1.1. In all experiments we used

the Casein Kinase-1 peptide as the substrate. The

specificity of this peptide greatly diminishes the chance

of phosphorylation by other kinases, a notion which

appears to be confirmed by the fact that the inhibition of

the recombinant TcCK1.1 exhibits a pattern almostidentical to that of the total T. cruzi extracts, indicating

that most of the activity measured in the latter was due

exclusively to CK1 and not to other kinases present in

the extracts. Another inhibitor of some CK1s, heparin, a

polyanion which serves to distinguish between the two

casein kinases, CK1 and CK2, was tested on recombi-

nant TcCK1.1. A low inhibition (30%) was obtained at

30 mg ml�1. This is comparable with data published formammalian CK1 with an IC50 of 24 mg ml�1 when

assayed at 2 mg ml�1 casein [38] while the IC50 of CK2

is 5/0.15 mg ml�1 [39]. Apart from this data, the

biochemical parameters of recombinant TcCK1.1 point

to a structural difference between T. cruzi CK1 and

most other CK1s reported. TcCK1.1 has one of the

highest reported Km values for ATP, with the exception

of those of rat liver nuclei and yeast [1]. As for b-casein,we calculated a Km higher than that of most other

organisms [7,25,40�/42]; however, when using the spe-

cific peptide Casein Kinase-1 substrate, our TcCK1.1

gave us a Km value somewhat lower than what was

calculated for other CK1s [25]. It should be mentioned

that the concentration of NaCl used in the assays for

CK1 vary from group to group. However, based on

studies carried out by Calabokis et al. [16] on theoptimal salt concentration for a partially purified CK1

of T. cruzi at a concentration of 150 mM, which is what

we used, the activity varies only 30% from the maximum

and 60% from the minimum possible without the

addition of any salts. Therefore, most of our compar-

isons are made with groups that used similar assay

conditions. Finally, the low activity of the inhibitor

CKI-7 denotes that the affinity for the nucleotidebinding site is lower than in other species [4,7]. Our

observations are confirmed by the work of Calabokis et

al. [16], with their reported values on the biochemical

parameters of a partially purified CK1 of T. cruzi

agreeing, for the most part, with those we have

obtained. Furthermore, Vancura et al. [40] worked

with a yeast homolog of CK1, YCK2 and found only

a 30% inhibition with concentrations up to 500 mM. Soit is not unusual to find some members of the family for

which the inhibitor does not respond efficiently. This is

another clue pointing to a structural difference of T.

cruzi CK1, making it all the more relevant when looking

for potential drug targets against the parasite.

As we mentioned before, a peculiarity of the CK1

genes characterized here is the fact that one of them is

arranged in tandem. In many organisms there have beenreports of CK1 present in various isoforms, and as

different splicing products, but in our case we have the

same isoform, TcCK1.2 , repeated several times. In

trypanosomatids, many housekeeping genes are present

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/36 33

in large tendem repeated clusters. These duplications

may be involved in the regulation of expression for these

genes. In fact, TcCK1.2 was more highly expressed than

TcCK1.1 . Comparative analysis of the TcCK1s mRNA

expression reflects a significant contribution from

TcCK1.1 in the infective stage. Otherwise, TcCK1.1

seems to contribute little to the general TcCK1s expres-

sion. We cannot rule out the possibility of another CK1

isoform contributing to the overall expression and

activity, particularly with respect to the mRNA expres-

sion of amastigotes where the increased signal seems to

be superior to that of the two isoforms we have isolated

together. The results of the CK1 activity assays in the

three life forms reveal that the two mammalian stages,

amastigotes and trypomastigotes, have a significantly

higher enzyme activity than the insect stage. Common to

these stages of the parasite is the need to adjust to

diverse physiological environments and cell require-

ments, such as temperature, host�/parasite signaling,

trafficking, etc., which might require the presence of

numerous metabolism regulators, such as CK1. The fact

that the CK1-like activity presents differences with

respect to the mRNA expression can be attributed to

post-translational modifications as proteolysis, phoryla-

tion, and localization of the protein that have been

reported for CK1 [43,44].

TcCK1.1 mRNA expression varies along the cell

cycle, while TcCK1.2 mRNA, after being released

from arrest, achieves a level which tends to remain

constitutively high. In spite of this constant mRNA

expression, a drop in the CK1-like activity is found in

the middle of the cell cycle, probably due to post-

translational regulations as mentioned above. The

results of CK1-like activity measurements in the syn-

chronized cultures show an increase in the enzyme

activity corresponding to the cell entry into S, confirmed

by [3H]Thymidine incorporation assays (not shown),

and probably M phases. The presence of a peak for a

CK1-like activity in mitosis confirms the earlier findings

of other groups [45,46], but still needs to be character-

ized further in T. cruzi . What was certainly unexpected

and new to us was the peak of activity found during the

phase of DNA synthesis, appearing consistently

throughout our assays. Substrates found for CK1 in

other organisms, and its action on them, also attest to

the probable function of CK1 in regulating DNA

replication, as is the case with the blockage of the origin

of replication of the SV40 large T antigen by CK1 [47].

Thus a probable role of TcCK1s in growth and cell cycle

control cannot be ruled out. This concept is further

supported by our studies of the CK1-like activity during

epimastigote growth, the results of which reveal some

ongoing activity even in cells with null or little replica-

tive activity, but with an evident increase in enzyme

activity during the log cell phase.

A possible tie between all our observations related to

TcCK1 function is the phosphorylation of structural

substrates. Tubulin, troponin, myosin, band 4.1, kine-

sin, tau, and flagellar dynein, all of them structure-

related proteins, have been described as possible CK1

substrates [2,48,49]. Some of them would need the

action of one isoform or the other, depending on the

life stage of the parasite. Dynein, apart from being

present in the flagella, is a microtubule motor protein

present in the mitotic spindles and centrosomes, and has

an important role in mitosis when microtubules lay

down the structure for chromosome segregation. CK1

has been linked to centrosomes and mitotic spindle [50],

most conspicuously following induced DNA damage,

through phosphorylation of the tumor suppressor

protein p53 [51], just as HRR25 is linked to DNA

repair in S. cerevisiae [12] and D. melanogaster CK1 is

induced by DNA perturbation [52]. Our TcCK1s could

play similar roles in the parasite. Not only do they have

a nuclear localization signal that would situate them

spatially suited to act during mitosis: they also have a

kinesin homology sequence, another reason to make one

ponder the possible relationship CK1 has with structural

proteins, especially those associated with microtubules.

Our results would not seem to rule out this possibility.

First, the subcellular fractions containing the most CK1-

like activity were those enriched with membranes, where

the cytoskeletal proteins remain. Second, the activity

also peaks during the S and M phases of the cell cycle,

especially in M, when the presence of microtubules is

vital. Moreover, apart from the synchronized cultures,

when the parasites are more actively replicating, that is,

in the middle of the logarithmic phase, the CK1-like

activity of T. cruzi reaches its peak. Third, the fact that

one isoform is repeated in tandem points to a high

requirement of CK1 activity, which is usually the case

when the substrates are structural proteins with large

needs for the enzyme. All of the above could lead us to

envision a future link between TcCK1s and cytoskeleton

proteins such as microtubules, microtubule associated

proteins (MAPs), or microtubule motor proteins like

kinesin and dynein.

CK1 has been described as a pleitropic enzyme,

meaning that it could be involved in metabolism,

motility, trafficking and/or signalling. The presence of

expression and activity of TcCK1 in the parasite,

regardless of the life stage in which it is found, and

with all the different physiological conditions that each

stage implies, seems to support this idea.The possibility of multifunctionality, together with

the finding that the sequences have a unique insertion

which most probably confers structural differences to

the TcCK1 isoforms with respect to other CK1s make

TcCK1s very relevant when possible molecular targets

are considered in the fight against Chagas disease. At

C. Spadafora et al. / Molecular & Biochemical Parasitology 124 (2002) 23�/3634

the same time, our results with T. cruzi may help to shed

new light on possible roles of CK1 in higher eukaryotes.

Acknowledgements

The authors would like to thank L. Meijer and G.R.

Pettit for kindly providing hymenialdisine. We thank

Pilar Navarro for help in parasite cultures. We thank N.

Galanti, I. Espinoza, J. Allende and V. Pulgar for

suggestions, help, and guidance in some experiments,

as well as G. Robledo for very useful collaboration. We

thank all our laboratory colleagues for helpful discus-

sions as this work progressed. C. Spadafora is recipientof a Becas 2003 fellowship from Secretarıa Nacional de

Ciencia y Tecnologıa (Panama). C. Torres is recipient of

a fellowship from the Fundacion Ramon Areces (Ma-

drid, Spain). This work was supported by The Spanish

Grants PM97-0139 (F. G.), PM98-0115 (S. Castanys),

CICYT-FEDER IFD97-0747-C04-03 (S. C.), the FON-

DECYT-Chile 1020095 (A. Morello), and the Convenio

CSIC-Universidad de Chile between F. Gamarro and A.Morello (1997�/2000).

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