Molecular and Biochemical Parasitology, 55 (1992) 127 134 © 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

127

MOLB10 01816

Cloning and characterization of the gene encoding Trypanosoma cruzi DNA topoisomerase II Stenio P. Fragoso and Samuel G o l d e n b e r g Fundafdo Oswaldo Cruz, Dept. Bioquimica e Biologia Molecular, Laborat6rio de Expressdo Gdnica, Rio de Janeiro, Brazil (Received 4 May 1992; accepted 18 June 1992)

The gene encoding Trypanosoma cruzi type I! topoisomerase (TcTOP2) was isolated from a genomic library with a heterologous probe corresponding to part of the Trypanosoma brucei type I1 topoisomerase (TBrTOP2) gene. Nucleotide sequencing of TcTOP2 showed that the gene consists of an open reading frame of 3696 nucleotides (1232 amino acids), predicting a polypeptide product of 138 413 Da. Comparison of the amino acid sequence with that of type II topoisomerases from T. brucei (TBrTOP2) and Crithidiafasciculata (CfaTOP2), shows a high degree of conservation with estimated identities of 78% and 69%, respectively. TcTOP2 is a single copy gene in the genome of T. cruzi Dm28c and is expressed as a 4.5-kb mRNA. PCR mapping showed two distinct mini-exon addition sites at positions 225 and 203 upstream from the initiator AUG. Key words: DNA topoisomerase II; Trypanosoma cruzi; Chagas' disease; Gene cloning

Introduction

DNA topoisomerases are widely distributed in nature, and catalyse conformational changes in DNA by breaking and rejoining DNA strands. In generic terms, these enzymes are classified according to their origin from eukaryotes or prokaryotes, and as type I or type II, depending upon their mode of cleaving DNA. Type I topoisomerases introduce tranCorrespondence address: Samuel Goldenberg, Fundag~o Oswaldo Cruz, Dept. Bioquimica e Biologia Molecular, Avenida Brasil 4365, Rio de Janeiro, R J, 21045, Brazil. Fax: 55-21-5903495. Note." Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M91165. Abbreviations: TcTop2, T. cruzi type II topoisomerase; kDNA, kinetoplast DNA; Topo II, type II topoisomerase; TBrTOP2, T. brucei type II topoisomerase; CfaTOP2, C. fasciculata type II topoisomerase; LIT, liver infusion tryptose; SDS, sodium dodecyl sulphate; Tris, 2-amino-2-hydroxymethylpropane-l,3diol; NP-40, Nonidet P-40.

sient single-strand cuts into duplex DNA molecules, while type II topoisomerases (Topo II), which in general require ATP, catalyse double-stranded cuts of the DNA (reviewed in refs. 1 and 2). These enzymes are involved in DNA metabolism (replication, transcription and recombination) and eukaryotic Topo II is also important for maintenance of chromosome structure [3-5] and chromosome segregation [6,7]. It is believed that DNA topoisomerases play an important role in the replication of the unusual kinetoplast DNA (kDNA) found in trypanosomatids [8-11]. This DNA is structured as a compact network formed by two distinct classes of interlocked circular DNA molecules, the maxicircles and the minicircles. The former encode normal mitochondrial gene products (reviewed in ref. 12), and the latter encode at least part of the guide RNAs necessary for RNA editing [13]. During kDNA replication the minicircles must be released from the network, and subsequently religated upon segregation to the daughter

128

cells, indicating the necessity for active participation of Topo II in this process. In addition, it is very likely that Topo II plays an important role in the organization of the kDNA network of trypanosomes whose differentiation is accompanied by changes in the conformation of the kinetoplast. This is the case in Trypanosoma cruzi where replicative forms (amastigotes and epimastigotes) present a compact (bar-shaped) kinetoplast, while infective forms (trypomastigotes) present a more relaxed kDNA organization (basket form) [14]. Accordingly, type II topoisomerase activities have been described in trypanosomatids [811,15,16], and at least in Crithidiafasciculata it was clearly demonstrated that the enzyme was localized in the kinetoplast [17]. In the case of T. cruzi, it was observed that inhibitors of bacterial type II topoisomerase are able to block the replication and the differentiation of the parasite, and ultrastructural data suggested that the kinetoplast was the target of the drugs [18]. On the other hand, a nuclear ATPindependent Topo II has been described in T. cruzi [15], suggesting that distinct Topo II activities might exist in this parasite. Recent work resulted in the cloning of the genes encoding Topo II from Trypanosoma brucei (TbrTOP2) [16] and from C. fasciculata (CfaTOP2) [19]. DNA sequence analysis showed that they share regions of homology to Topo Il of other eukaryotes. However, the Crithidia enzyme was purified from the kinetoplast suggesting that in this organism the same gene might encode the nuclear and the mitochondrial forms of the enzyme. The observation that inhibitors of bacterial Topo II block replication and differentiation of T. cruzi and that the C. fasciculata Topo II, although displaying eukaryotic Topo II features, is located in the kinetoplast [17], suggests that the trypanosomatid enzyme might present special features that would render this enzyme a good target for the chemotherapy of diseases caused by trypanosomatids. In this article we report the cloning and sequencing of the gene encoding T. cruzi Topo II, and we compare its sequence homology with the genes isolated from T. brucei and from C. fasciculata.

Materials and Methods

Nucleic acids isolation. Nucleic acids were isolated from T. cruzi Dm28c [20] epimastigotes. Parasites in late log growth phase were harvested from LIT medium [21], lysed with 0.5% NP-40 under isotonic conditions [22] and the nuclei were partially purified through three rounds of centrifugation at 1000 x g at 4°C. This fraction was digested with RNase free of DNase for 2 h at 37°C and further digested with proteinase-K for 12 h in 10 mM NaC1/5 mM EDTA/0.5% SDS/10 mM Tris-HCl, pH 7.6. The DNA was phenol extracted and extensively dialysed against 100 mM NaC1/1 mM EDTA/10 mM Tris-HC1 pH 7.6. Poly(A) + RNA from epimastigote forms was obtained by oligo-dT cellulose chromatography as described elsewhere [22]. Construction and screening of genornic libraries. T. cruzi genomic DNA was partially digested with Sau3A, and fragments in the size range of 12-20 kb were isolated by NaC1 density gradient centrifugation and ligated to BamHI-digested EMBL-3 and EMBL-4 (Promega Corp., Madison, WI) [23]. The ligated DNA was amplified in E. coli LE392 after packaging with Packagene extract (Promega Corp., Madison, WI), and the resulting library contained 5 x l0 s recombinant clones. The genomic libraries were then screened with a 2.7-kb Smal fragment from recombinant plasmid pDC806 (De Cicco and Englund, unpublished) (TbrTOP2Sma), which was radioactively labelled by nick translation [24], and contains part of the coding region of T. brucei topoisomerase II gene. The filters containing the recombinant phages were hybridised overnight at 65°C in 5 x SSC (1 x SSC is 150 mM sodium chloride/15 mM sodium citrate)/5 x Denhardt's solution (1 x Denhardt's solution is 0.02% polyvinyl pyrrolidone/0.02% Ficoll/0.02% bovine serum albumin)/0.1% SDS/100 #g m1-1 sonicated salmon sperm DNA, and then washed twice in 2 x SSC/0.01% SDS, twice in 1 x SSC/ 0.01% SDS and once in 0.5 x SSC/0.01% SDS at 65°C. The positive clones were purified

129 according to standard protocols [24] and analyzed by Southern blotting [25] after digestion with the restriction endonucleases EcoRI, Sail, BamHI, PstI and HindlII, using the hybridization conditions described above. DNA sequencing and computer analysis. DNA restriction fragments of the gene encoding TcTOP2 were sub-cloned in the vectors M 13 mp 18 and mp 19 [26] and sequenced using the dideoxy chain termination method [27] with T7 DNA polymerase (Sequenase, United States Biochemical Corp., USA). Some M13 clones were sequenced using the ExolII/S1 nuclease strategy [28]. The sequences were analyzed using the programs of the University of Wisconsin GCG software package [29]. Cloning of the 5'-non coding region of TcTOP2 mRNA. The 5' end of the TcTOP2 mRNA was obtained using two oligonucleotides corresponding to 20 nucleotides of the T. cruzi mini-exon sequence (ACAGTTTCTGTACTATATTG) [30] and 18 nucleotides of the 5'-coding region of TcTOP2 (GGCGTTGAGGAGAATTTC), respectively. These primers were used to amplify a cDNA library of T. cruzi in 2gtll using PCR [31] with Taq DNA polymerase (Promega Corp., Madison, WI). The cDNA library was constructed using random primers to prime the first strand synthesis [32] followed by alkaline hydrolysis and priming of the second strand synthesis with the spliced-leader oligonucleotide described above. The cloning in 2gtll followed standard protocols [33]. The oligomacleotides used for PCR amplification contained in their 5'-end the EcoRI recognition sequence GGGAATTC, in such a way that the PCR products could be directly cloned in M 13mpl 8 after EcoRI digestion. Southern blot analysis. Genomic DNA from T. cruzi (5/~g) was digested to completion with the restriction endonucleases EcoRI, Sail, BamHI, HindlII, XhoI and PstI (New England Biolabs, Boston, MA), electrophoresed in a 0.8% agarose gel in TBE buffer (89 mM Tris base/89 mM boric acid/2 mM EDTA) and

transferred to a nylon membrane (Hybond, Amersham, UK) using a vacuum blotting system (VacuGene XL, Pharmacia, Sweden). The filter was hybridized overnight with the radiolabeUed probe at 65°C in 5 x SSC/5 x Denhardt's solution/0.01% SDS/100 pg m l salmon sperm DNA, and then washed twice in 2 x SSC/0.01% SDS, twicein 1 x SSC/0.01% SDS, once in 0.5 x SSC/0.01% SDS, and once in 0.1 x SSC/0.01% SDS, at 65°C.

Results and Discussion

Cloning and sequencing of T. cruzi topoisomerase H gene. Using a probe corresponding to part of the coding sequence of the T. brucei topoisomerase II gene (TbrTOP2Sma) a clone (2tcl) was selected from a previously amplified T. cruzi genomic library in EMBL-4 (containing 15 000 clones). Digestion of this clone with EcoRI released a 3.5-kb fragment that hybridised to TbrTOP2Sma, while digestion of this EcoRI fragment with PstI resulted in two fragments of 0.84 kb and 2.7 kb, of these, only the 0.84-kb fragment hybridized to TbrTOP2Sma. DNA sequencing of these fragments and comparison with the sequence of TbrTOP2 [16], showed that they corresponded to the 3'-end of the T. cruzi topoisomerase II gene (TcTOP2) (Fig. 1): the 0.84-kb fragment contained the 3'-coding region of the gene while the 2.7-kb fragment contained the 3'-non coding region of the gene. In order to determine the full sequence of the T. cruzi topoisomerase II gene, we screened an additional 40 000 clones from the EMBL-4 genomic library and 15 000 clones from a nonamplified EMBL3 genomic library, using the 0.84-kb EcoRI-PstI fragment. This resulted in the selection of two clones from the EMBL4 genomic library (2tc2 and 2tc3), and six clones from the EMBL3 genomic library (2tc4 to 2tc9). These clones were mapped with different restriction enzymes, and clones 2tc2 and 2tc5 were selected for sequencing. The partial restriction map of clones 2tc2 and 2tc5 is shown in Fig. 1. The DNA of clone 2tc2 was doubly digested with EcoRI and PstI-and

130

i f

XHS

T TT!

P

B

T ~

S

E

E

T

H

!

D

-_._,,. ~

B

Fig. I. Partial restriction map of TcTOP2 genomic clones and DNA sequencing strategy, partial restriction map of clones (A) 2Tcl, (B) 2Tc2 and (C) 2Tc5. The arrows below the figures indicate the TcTOP2 open reading frame. The EcoRl(e) and Sa/I(s) sites indicated at the extremities originate from the cloning vector. (D) consensus restriction map. (E) Restriction map and sequencing strategy of TcTOP2: Bold line indicates the open reading frame and thin lines the non-coding regions. EcoRl (E and e), Sall (S and s), PstI (P), BamHI (B), Hindlll (H), NarI (N), Sphl (SP), DraI (D) and Xhol (X).

probed in a Southern blot with the 0.84-kb fragment. The results showed that a 3.2-kb fragment hybridized to this probe. DNA sequencing of this fragment (Fig. 1) and comparison to TbrTOP2 showed that it corresponded to the coding region of the gene. However, 110 nucleotides were still missing from the 5'-coding region. This region, as well as the 5'-upstream region of the gene, were obtained from a 3-kb SalIEcoRI fragment of clone )~tc5 screened from the EMBL3 genomic library (Fig. 1). The DNA sequencing strategy is shown in Fig. 1. The results show that TcTOP2 gene has a long open reading frame of 3696 nucleotides which would encode a 138-kDa polypeptide (Fig. 2). This size is compatible with those described for TbrTop2 (137 kDa) [16] and for CfaTop2 (138 kDa) [19]. Organization of T. cruzi topoisomerase H gene. Southern blot analysis of T. cruzi genomic DNA with the 3.2-kb fragment from clone 2tc2 (Fig. 3) suggests that TcTOP2 is a single copy gene in the T. cruzi Dm28c genome, as is the case for TbTOP2 [16] and CfaTOP2 [19]. Digestion of genomic D N A with EcoRI and PstI generates fragments of 8.0 kb and 4.8 kb, respectively, (Fig. 3) that have sizes compatible with those of the 8.3 kb and 5.0 kb fragments observed upon digestion of the genomic clones

with the same enzymes (Fig. 1). Digestion of genomic DNA with SalI gives rise to three fragments of 13, 3.4 and 1.2 kb (Fig. 3). Two of these, 3.4 and 1.2 kb, can be predicted from the restriction map of the isolated clones, while the larger fragment was not present in the genomic clones analysed. The enzymes HindIII and XhoI, which cut once within the gene, release two fragments of 8.3 and 5.1 kb and 20.7 and 3.7 kb, respectively, supporting the assumption that TcTOP2 is a single copy gene. Using Northern blot analysis, a single poly(A) + RNA of 4.5 kb is observed when the filter is probed with the EcoRI-PstI fragment of 3.2 kb, originating from clone 2tc2 (data not shown). Mature trypanosomatid mRNAs contain a ubiquitous sequence, the mini-exon or spliced-leader, that is added posttranscriptionally by trans-splicing to the 5'-end of the primary m R N A transcript [34-36]. In order to determine the mini-exon addition site, a 450-bp fragment was amplified by PCR from a T. cruzi cDNA library using oligonucleotides corresponding to the mini-exon and the 5'coding region of the gene TcTOP2, respectively. This fragment was cloned in M13mpl8 and, surprisingly, two different clones (Topcrl and Topcr2) were obtained, which differed in their mini-exon addition site (Fig. 4). In the clone Topcrl the mini-exon addition site is located 225 nucleotides upstream from the

131 1 AAAAAATATATATTTGACGtGTTTGTGTT~GGGAAAAACAGAGAAGAAGAGGGGAGGGG~AGCGA~TG~cGi~G~CA~C~AG~A~AGPA~TC~CC~ 100 101 C~TC~Ac~At~T~T~TA~GG~G~AG~tG~AC~TT~GC~GC~TGDGAT~CA~C~C~T~CC~TG~Tf~TC~TT~AT~AT~AG~AG~C~GC~T~T~ 200

201 ~O~pOp~T~,~TC~%~C~T~p~T~Wp'~tT~T~T~,C~CC~C~C~T~^~C~'~8%%O~p~O

~

301 ^~T~P~tT~A~T~P°~cTPP~T~t~P~W8~8~%~T~T~c~fTT~t~P~°~PAPAWP~TTP~T~c 401 A p ~ T ~ T t W 8 % ~ e ~ T T F T C ~ C ~ % ~ C ~ ^ ~ p ~ W ~ % ~ ^ T ~ % T ~ p ~ p T W ~ O ~ T p p ~ T ~ p O ~ f

300 400 500

501 ~TT~TG~CT~AC~GCFTT~CC~TC~GC~GC~GC~CA~A~GG~AA~GFtT~AT~TG~G~GG~C~AT~AT~TG~GC~G~CG~CA~Cc~CA~GT~TG ~ 600 601 cGAACGTCG~CCCAAAAGA~AAGAATCTC~CACGGGTGAAGTTTCTTCC~GACTACGAACGCTTTGGAT~GGACGCGAA~AAAATCTCACACGACATGA~ 700 N V D P K E K N L T R V K F L P D Y E R F G L D A N K I S H D H K ~1 G~GT~TG~T~AC~AG~GC~TC~TG~AT~T~CC~CG~T~TT~GC~TT~AA~TA~A~TG~AC~G~TC~CG~TT~CC~TT~AA~CC~TT~CT~AC 800 801 ~A~CC~TG~TG~C~CT~CC~CC~GT~C~GT~GA~AA~TG~CA~CA~C~CG~TT~T~C~G~GC~GC~AT~GT~C~TT~CG~T~TT~CC~CC~ 900 901 TT~CC~CC~C~CA~GT~G~TC~TT~GC~TC~TG~AT~c~TT~TG~C~AC~AC~GC~GT~CC~AC~C~AC~CG~C~AG~G~TCITA~A~C~c 1000 1001 ACTGGAGAG~GTTGAGA~G~CACTGAAGA~GGAT~ACAA~TGATTGACACAAATCGTGtTTTGCGACA~TTC~TGATCCTTGTTTTTC~CGTGCAGGT~ 1100 L E S V E K A L K K D N K V ] D T N R V L R H F H ] L V F L V Q V 1101 ~AA~CG1~AG~TT~AT~CG~G~AC1~AG~CG~GC~TT~TT~CT~TG~C~C~TG~CG~G~TT~CA~GA~AG~AA~TG~G~AC~TT~TT~TT~GC~TG~ 1200 1201 ~G~TT~TCPG~CG~T~TC~AC~CC~TG~CG~GG~AG~C~CA~AT~AA~TT~AT~AG~AG~TG~GC~CC~GG~G~GC~TG~GC~GC~A~CT~TC~ 1300 1301 C~CT~CC~Tc~CG~AA~TG~TG~AC~CC~CC~CG~CT~G~GG~C~CA~GG~TC~TC~C~C~TC~T~TC~CG~AG~GC~AC~CT~CA~AG~C~TC1400 1401 G~CAGAA~fC~CTGTCAA~C~CCAGAAACGCTACACA~GTGTGTTCCCACT~CGTGG~AAGCTTTTAAACGTGCGCAATAAAAATCTCAAACGTCTT~ 1500 A Q N S L S S D O K R Y T G V F P L R G ~ L L N V R N ~ N L K R L ~

15Ol ^ ~ U ~ ^ P ~ ^ 8 A ~ P ~ T ~ T ~ ° T ~ T T P ~ P ~ P P ~ T ~ P ~ P P ` P ~ F ~ c P T ~ P ~ T ~ T ~

16oo

1601 C~TG~C~A~8AG~AT~CC~AT~GA~CA~AC~TC~AA~GA~TT~TC~TC~T~CC~TT~G~C~TA~G~CC~CG~TG~TG~AT~GC~T~C~GC~T~1700

17Ol ~Tq~TT~Tqc~°~T~T^P~T~T~T~P~^~P~c~%^T~c~TT~T~T~T~P~P~W~^~P~%A~P~%

1~oo

1801 ATGGAAATG~TTC~TACA~CGC~AAGTATfACAAGG~T~tCGGTA~AT~CA~AACAG~C~AGGGGAAGGAGTAcTTTAAAGA~ATGGATAAGCA~ACAAt 1900 G N V S Y T A E Y Y E A L G T S T T A E G E E Y F K D N D K H T H 1901 G~GA~TT~T~TG~AA~GT~AT~AC~AT~A~UA~TT~Ac~GT~TG~TT~AT~CC~AG~AA~TG~AG~GG~GC~AG~AT~GG~TG~CA~AG~C~AC~CT 2000 2001 ~AC~CA~GG~AA~TT~AC~AT~G~Gt~G~AG~CA~TG~CC~TA~CC~AT~TT~Tt~AC~AA~AG~TG~TG~AC~T~A~TT~Cc~GT~AT~CC~2100 2101 GC~CC~TT~cA~AC~CT~T~AT~GA~TA~AA~CC~CC~AA~GA~AA~T~TG~GG~CC~TT~TG~GT~C~CT~GT~Ac~AA~CG~CC~AA~TG~CT~2200 2201 GTTGTCAGG~TATATTTCG~AGGTTTCGGCCTTTC~CCACGGGGAAATGtCATTGCAAG~AACGATTATtAAAATGGCT~AAAATTTTACGGGTGGGAAt 2300 L S G Y I S E V S A F H H G E H S L Q E T I I K H A Q N F T G G N 2301 ~C~TT~AC~TT~TC~TT~AG~GT~TT~G~C~GT~AG~AG~T~GT~AT~t~AC~CG~CA~CT~GT~AC~C~TT~CA~A~TC~CC~GT[ 2400 2401 TG~CA~GG~A~TT~TC~C~GT~AA~AT~AG~CT~TG~G~A~AC~T~CG~AA~AA~GA~AG~AG~G~AG~CA~A~AC~AC~TT~CC~TT~TT~c2500 2501 T~TG~TG~T~GC~C~GC~GT~TT~GT~T~GA~TT~G~TT~CA~CAPC~TT~CT~cT~TT~AT~C~TT~AC~TT~C~CA~CG~t~G~GC~T~ 2600 2601 ATCAATGGC~AGGCTGCCA~AGTTGTAGTA~GGCGGCTT~TGCCGTGGGCTGTAGGATAtCAGGGTGAA~TACGTCGAG~GCCCGAAGG~GAATTTATT~ 2700 ] N G E A A K V V V R R L V P W A V G Y Q G E V R R G P E G E F ] A 2~1 CG~CC~GA~T~AT~AG~Ac~AC~T~AT~GC~GT~TG~AC~TG~CG~AG~TA~C~GG~C~TT~GC~T~AG~CA~TC~GC~AC~T~TT~CC~TT~T2800 2801 T~CT~CG~A~T~TT~TG~AG~GT~TT~cC~AC~AT~C~GT~CG~A~AT~TG~AC~TT~CUG~AA~TT~CG~AT~GC~CC~TG~CA~CG~AC~CG2900 2901 ~AG~GT~G~SGT~A~ETT~cCETC~CC~GT~TT~AC~TC~AT~GG~CT~TC~TC~Cc~CC~CT~GT~TC~TC~CC~CT~TG~AA~G~T~TG~CA~3000 3001 ~T~T~TTA~AGT~G~ATTACGA~G~G~CTTGA~TTTACAA~AAA~GA~GT~AA~TAA~TT~G~TCTT~AG~f~AA~T~G~Ac~GAGAAATC 3100 V L Q ~ H Y D R R L D L Y E E R R Q R N L G L L E A E L A R E E S

31Ol ~T~p~W~T~%A~Wf~pApp~TtW~TTUp~F^~W~T~T~pp~F,~T~TpA~OTpU 32Ol ~ % T ~ W p T p p T ~ p ~ T ~ O ~ p ^ ~ F ~ T T ~ p p ^ ~ O ~ F ~ p O ~ T p ~ T ~ p ~ T ~ p ^ ~ p ~

3~o0 ~

3300

33Ol AAi~c~Tc~c~Tc~^~A~c~c~cT~TT~A~it~TT~^~Tc~Ac~c~Tc~c~c~c~TT~A~T~A~c~c~c~t

3400

34Ol ~ ^ ~ ^ T t ~ T T ^ ^ ~ c ~ c ~ T ~ c ~ ^ ^ ~ A ~ A T ~ T T A ~ T ~ ° ~ ^ A ~ t ~ ` ~ T ~ ° A ~ t T ~ ^ ~ ° ^ ° ° ~ ^ ~ T c T ~

3500

A T I L K E R R V N P P T G D V $ R N L O O P R L E L E E V K V S

3501 ~ c T ~ c c ~ ^ ^ ~ c ~ t c ~ c ~ T ~ i ~ T ~ T ~ ^ ~ P c ~ f ~ c c ~ c ~ c ~ c ~ T ~ ^ ^ ~ c c ~ A T ~ t ~ c ~ t ~ T T ~ T

~

37Ol ~U~AT~T~cAP~A~T~cAi~c~Tc~TT~A~A~T~c~TT[T~tT~A~AcAT~c~ccAATT~AA~^~AtT~AA^~AAAA

3600

3~oo

3801 A~G~A~GGeG~GGcATTT~TAAccA~cT~AAGT~AAAAAAAAA~AAAAGGG~GG~GG~G~GGA~AGAAGcT~tGAAccTAG~TGAGTcT~G~ 3900

3901 GATGGGGTG~GCTCATCATtACATGATAGAGCATAGAAGtTTTCCTTTTAAATCCGCCG~AATGAAAAAtAAAATAA 3977

Fig. 2. Nucleotide and predicted amino acid sequence of TcTOP2.

A U G c o d o n while in clone Topcr2 it is located at position 203. Furthermore, clone Topcrl

contains two adenine residues and one guanine residue at positions - 165, - 164 and -- 65,

132

!: p $ 8

# X -245 -195 -145 -95 -45 6 56

Kbp 23.1 +

9.4-

TOPCRI A C A G T T T C T G T A C T R T A T T G (~CATCATAAT CCGCGGGCGC AACCAAAAAA A A A A A A A ~ G C C & T C C T T ~ CACI%TAGACA T A C G C A C A C A AGTTTTATTG TCGTTTTAAA TATATTTA~ GTTTGTGTTG GGGAAAAACA GAGAAGAAGA GGAGGCATCG AAGTACAAGA AACTCACCCC GGCCGGAGAT GTACGTTGGC AGCGT~ATA

TTGAACGACA G~I%AARAGG TTTGTGCGCA AAAAAATATA G~OAGGGGG CATCGACCAT CATCCTCCAG

G~AGCAGCTA AAAGAGACCC TAGACCACGG TATTTGACGT AGCGAATGOC GTCCTCATAC TTCCATGTTT

106

G T C T T T G A T C A T G A G A & G G G C C G C A T G G T G TGGG~%GTCAC T G A A G G T C A A

156

CCACGGCCTC CTGAAGATCG TGGATGAAAT TCTCCTCAAC GCC TOPCR2

8

6.6+

4.4. D

2.3~

2.0+

0.56+

Fig. 3. Southern blot analysis of T. cruzi Dm28c genomic DNA. Genomic D N A (5 ~Lg)was digested with EcoRI (E), Pstl (P), Sall (S), BamHI (B), HindII1 (H) and Xhol (X) and electrophoresed in a 0.8% agarose gel. Following transfer to a nylon membrane, the immobilised DNA was probed with a 3.2-kb EcoRl/Pstl fragment from clone 2Tc2. The 1.2-kb SalI fragment is indicated by an asterisk. The position of molecular weight markers is indicated on the left of the figure.

respectively, (Fig. 4) that are not present in clone Topcr2. Whether these differences are biologically meaningful or merely an artefact arising from PCR, remains to be determined. However, it is interesting to note that distinct mini-exon addition sites have been also observed in the case of CfaTOP2 m R N A (Dan Ray, personal communication). Comparison of TcTOP2 with TbrTOP2 and

-222 -172 -122 -72 -22 29 79 129 179

ACAGTTTCTG TACTATATTG Ia.AAAAAAGAAAAARAGGAI~ GCACACATTT GTI3~_ ~ T A G A T T T A A R A R A ARATAT~%TAT AAGAAGA6~G GAGGGGOAGC TCACCCCCAT CGACCATGTC GTGGATACAT CCTCCAGTTC CATGGTGT~G GAGTCACTGA ATGAAATTCT CCTCAACGCC

GAGCAQCTAC CGCGGGCGCA RCCAAAAAAA GAGACCCGCC ATCCTTACAC ATAGACATAC ACCACGGAGT TTTATTOTCG "a.-I-JL-J:AAATAT TTGACGTGTT TGTGTTGGGO RAAAACAGAG GAA.___~CGGA G G ~ T C G A A G T A C A A G A A A C C T C A T A C G G C CGG/tGATGTA C G T T G G C A G C CATGTTTGTC TTTGATCATG AGAAGGGCCG AGGTCAACCR CG~CCTCCTG AAGATCGTGG

Fig. 4. Nucleotide sequence of clones Topcrl and Topcr2. The oligonucleotides used to amplify the fragments by PCR and the initiator ATG codon are underlined. Nucleotide residues that differ between the clones are included in boxes.

CfaTOP2. Comparison of the predicted amino acid sequences of the three trypanosomatid topoisomerase II genes sequenced so far shows that TcTOP2 displays a 78% identity and 87% similarity to TbrTOP2 [16] and 69% identity and 80% similarity to Cfatop2 [19]. The alignment of the sequences indicates that the carboxy-terminal portion of the enzyme is the less conserved region, and that TcTOP2 is more homologous to TbrTOP2 than to CfaTOP2 (data not shown). The possibility that TcTOP2 is located in the kinetoplast, as is the case for CfaTOP2 [17], is of great interest in view of the potential role of this enzyme in the replication and conformational changes undergone by the kDNA during the life-cycle of T. cruzi. Recent results from our laboratory showed that inhibitors of bacterial type II topoisomerase are able to block the replication and differentiation of the parasite, and ultrastructural analysis of parasites indicated that the kinetoplast was the target organelle for the drugs tested [18]. Whether TcTOP2 is the target of these inhibitors remains to be determined, since this enzyme is more closely related to eukaryotic (30-35% homology) than to prokaryotic type II topoisomerase (19% homology to E. coli

133

gyrase A). On the other hand, the presence of a bacterial type topoisomerase in the kinetoplast of T. cruzi cannot be excluded. The overexpression of TcTOP2 gene will allow the study of enzyme structure and kinetics. It is worth mentioning that an ATP-independent topoisomerase II, with a molecular weight of about 200 000, has been isolated from T. cruzi [15]. This estimated molecular weight differs from that deduced for TcTOP2 (138 kDa), while this latter estimation is in agreement with the values obtained for TbrTOP2 [16] and CfaTOP2 [19]. In addition, TcTOP2 is very likely ATP-dependent in view of its homology to CfaTOP2, which was first purified as the enzyme that catalyzes the ATP-dependent decatenation of minicircles from the kDNA network [10]. This suggests that T. cruzi might express different topoisomerase II activities. We are presently raising an antiserum against recombinant TcTOP2 in order to carry out the immunolocalization of this enzyme in T. cruzi.

Acknowledgements We thank Drs. Paul Englund and Diana de Cicco for kindly providing plasmid pDC806 that contained part of the coding sequence of T. brucei topoisomerase II gene. We are obliged to Paul Englund for his interest in this work and helpful suggestions to the preparation of this manuscript. Catherine Lowndes is acknowledged for the critical reading of this manuscript, and Armindo Caldeira for his technical support. This investigation received financial support from the UNDP/WORLD BANK/WHIO Special Programme for Research and Training in Tropical Diseases and from Conselho Nacional do Desenvolvimento Cientifico e Tecnol6gico.

References 1 Vosberg, H.-P. (1985) DNA topoisomerases: enzymes that control DNA conformation. Curr. Top. Microbiol. Immunol. 114, 19-102. 2 Wang, J.C. (1985) DNA topoisomerases. Annu. Rev.

Biochem. 54, 665-697. 3 Earnshaw, W.C., Halligan, B., Cooke, C.A., Heck, M.M.S. and Liu, L.F. (1985) Topoisomerase II is a structural component of mitotic chromosome scaffolds. J. Cell Biol. 100, 1706-1715. 4 Gasser, S.M., Laroche, T. Falquet, J., Boy de La Tour, E. and Laemmli, U.K. (1986) Metaphase chromosome structure: involvement of topoisomerase II. J. Mol. Biol. 188, 613~29. 5 Adachi, Y., Luke, M. and Laemmli, U.K. (1991) Chromosome assembly 'in vitro': topoisomerase II is required for condensation. Cell 64, 137-148. 6 Dinardo, S., Voelkel, K. and Sternglanz, R. (1984) DNA topoisomerase II mutant of Saccharomyces cerevisae: topoisomerase II is required for the segregation of daughter molecules of the termination of DNA replication. Proc. Natl. Acad. Sci. USA 81, 2616-2620. 7 Holm, C., Goto, T., Wang, J.C. and Botstein, D. (1985) DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41,553-563. 8 Shlomai, J. and Zadok, A. (1983) Reversible decatenation of kinetoplast DNA by a DNA topoisomerase from trypanosomatids Nucleic Acids Res. 11, 4019-4030. 9 Chakraborty, A.K. and Majumder, H.K. (1987) Decatenation of kinetoplast DNA by an ATP-dependent DNA topoisomerase from the kinetoplast hemoflagellate Leishmania donovani. Mol. Biochem. Parasitol. 26, 215-224. 10 Melendy, T. and Ray, D.S. (1989) Novobiocin affinity purification of mitochondrial type II topoisomerase from trypanosomatid Crithidia fasciculata. J. Biol. Chem. 264, 1870--1876. 11 Shapiro, T.A., Klein, V.A. and Englund, P.T. (1989) Drug-promoted cleavage of kinetoplast DNA minicircles. J. Biol. Chem. 264, 4173-4178. 12 Simpson, L. (1986) Kinetoplast DNA in trypanosomatid flagellates. Int. Rev. Cytol. 99, 119-179. 13 Sturm, N.R. and Simpson, L. (1990) Kinetoplast DNA minicircles encode guide RNAs for editing of cytochrome oxidase subunit III mRNA. Cell 61,879-884. 14 De Souza, W. (1984) Cell biology of Trypanosoma cruzi. Int. Rev. Cytol. 86, 197-283. 15 Douc-Rasy, S., Kayser, A., Riou, J-F. and Riou, G. (1986) ATP-independent type II topoisomerase from trypanosomes. Proc. Natl. Acad. Sci. USA 83, 71527156. 16 Strauss, P.R. and Wang, J.C. (1990) The TOP2 gene of Trypanosorna brucei: a single copy gene that shares extensive homology with other TOP2 genes encoding eukaryotic DNA topoisomerase II. Mol. Biochem. Parasitol. 38, 141-150. 17 Melendy, T., Sheline, C. and Ray, D.S. (1988) Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidiafasciculata. Cell 55, 1083-1088. 18 Gonzales-Perdomo, M., De Castro, S.L., Meirelles, M.N.S.L. and Goldenberg, S. (1990) Trypanosoma cruzi proliferation and differentiation are blocked by topoisomerase II inhibitors. Antimicrob. Agents Chemother. 34, 1707-1714. 19 Pasion, S.G., Hines, J.C., Aebersold, R. and Ray, D.S. (1992) Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata. Mol. Biochem. Parasitol. 50, 57-68.

134 20 Contreras, V.T., Araujo-Jorge, T.C., Bonaldo, M.C., Thomas, N., Barbosa, H.S., Meirelles, M.N.S.L. and Goldenberg, S. (1988) Biological aspects of the Dm28c clone of Trypanosorna cruzi after metacyclogenesis in chemically defined medium. Mere. Inst. Oswaldo Cruz 83, 123 133. 21 Camargo, E.P. (1964) Growth and differentiation of Trypanosoma cruzi. Origin of metacyclic trypanosomes in liquid media. Rev. Inst. Med. Trop. $5.o Paulo 6, 93 100. 22 Goldenberg, S., Salles, J.M., Contreras, V.T., Lima Franco, M.P.A., Katzin, A.M., Colli, W. and Morel, C.M. (1985) Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi. FEBS Lett. 180, 265-270. 23 Kayser, K. and Murray, N.E. (1985) The use of phage lambda Replacement vectors in the construction of representative genomic DNA libraries. In: DNA Cloning: A Practical Approach, Vol. I., (Glover, D.M., ed.), pp. 1-47, IRL Press, Oxford. 24 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 25 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. 26 Norrander, J. Kempe, T. and Messing, J. (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26, 101106. 27 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 28 Henikoff, S. (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351 359. 29 Devereux, J., Haeberli, P. and Smithies, O. (1984) A

comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387 395. 30 McCarthy-Burke, C., Taylor, Z.A. and Buck, G.A. (1989) Characterization of the spliced leader genes and transcripts in Trypanosoma cruzi. Gene 82, 177 189. 31 Tung, J.-S., Daugherty, B.L., O'Neil, L., Law, S.W., Han, J. and Mark, G.E. (1989) PCR amplification of specific sequences from a cDNA library. In: PCR Technology: Principles and Applications for DNA Amplification (Erlich, H.A., ed.), pp. 99 104. Stockton Press, New York. 32 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1987) Current Protocols in Molecular Biology. Wiley, New York. 33 Huynh, T.V., Young, R.A. and Davis, R.W. (1985) In: DNA Cloning. A Practical Approach, Vol. I (Glover, D.M., ed.), pp. 49 78. IRL Press, Oxford. 34 Boothroyd, J.C. and Cross, G.A.M. (1982) Transcripts coding for variant surface glycoproteins of Trypanosoma brucei have a short, identical exon at their 5' end. Gene 20, 218-289. 35 Van der Ploeg, L.H.T., Liu, A.Y.C., Michels, P.A.M., De Lange, T., Borst, P., Majumder, H.K., Weber, H., Weeneman, G.H. and Van Boom, J. (1982) RNA splicing is required to make the messenger RNA for a variant surface antigen in trypanosomes. Nucleic Acids Res. 10, 3591-3604. 36 Perry, K., Watkins, K. and Agabian, N. (1987) Trypanosome mRNAs have unusual 'Cap 4' structures acquired by addition of a spliced leader. Proc. Natl. Acad. Sci. USA 84, 8190-8194. 37 Worland, S. and Wang, J.C. (1989) Inducible over expression, purification and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae. J. Biol. Chem. 264, 4412-4416.