Cloning and sequencing of the Leishmania major actin-encoding gene

Cloning and sequencing of the Leishmania major actin-encoding gene

Gene, 139 (1994) 123-125 0 1994 Elsevier Science B.V. All rights reserved. GENE 123 0378-l 119/94/$07.00 07693 Cloning and sequencing of the Leis...

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Gene, 139 (1994) 123-125 0 1994 Elsevier Science B.V. All rights reserved.

GENE

123

0378-l 119/94/$07.00

07693

Cloning and sequencing of the Leishmania major actin-encoding gene (Cytoskeleton;

Monika Whitehead

polymerase

chain reaction;

recombinant

DNA; sequence

homology;

Trypanosomatids)

V. de Arruda and Paul Matsudaira Institute for Biomedical Research, and Department

MA 02142, USA. Tel. (l-617) Received by G.N. Godson:

258-5188: Fax (l-617)

of Biology, Massachusetts

Institute of Technology.

Nine Cambridge Center, Cambridge,

258-7663

1June 1993; Revised/Accepted:

24 September/4

October

1993; Received at publishers:

8 November

1993

SUMMARY

We report Leishmania

the cloning

and characterization of the genomic sequence of the actin (Act)-encoding gene (act) from maps of two genomic clones, as well as genomic Southern analysis strongly suggest that

major. Restriction

the act of L. major is a single-copy gene. A single 1.6-kb transcript is detected in Northern acid sequence shows 68%89% identity with Act sequences from other eukaryotes.

fungi, protozoa served proteins.

INTRODUCTION

Leishmanias

somatid

are members protozoan parasites

of a large group of trypanothat are the causative agents

of a wide spectrum of human diseases. Their life cycle displays both an extracellular and an intracellular phase. During the extracellular phase they develop in the digestive tract of sand flies, as a flagellate promastigote. The intracellular phase of development occurs inside vertebrate macrophages, as an aflagellate amastigote. Throughout the differentiation process the parasites undergo a dramatic change in their morphology which must reflect rearrangement in their cytoskeleton. In eukaryotes, actin (Act) is the major component of the cytoskeleton and plays an essential role in a large number of cellular events, such as cell motility, cytokinesis, phagocytosis, etc. Actin is ubiquitous in the plants,

Correspondence to: Dr. M. V. de Arruda at her present address: BASF Bioresearch Corporation, 100 Research Drive, Worcester, MA 01605-4314, USA. Tel. (l-508) 849-2620; Fax (l-508) 754-7784. Abbreviations: aa. amino acid(s); Act, actin( act, gene encoding or 1000 bp; L., actin; bp, base pair(s); H., Herpetomonas ; kb, kilobase Leishmania; Mb, lo6 bp; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction: pfu, plaque-forming unit(s); T., Trypanosoma. SSDI 0378-l 119(93)E0617-M

blots. The deduced

amino-

(Trypanosoma

and animals, and is one of the most conThe existence of Act in trypanosomatids cruzi, T. brucei, Leishmania mexicana and

Herpetomonas

samuelpessoai)

has been determined

by in-

direct immunofluorescence and immunoblots using heterologous anti-Act antibodies (de Souza et al., 1985; Mortara,

1989) and by isolation

Amar et al., 1988). Little is known about

of the act gene (Ben

the role of Act in the life cycle

of the trypanosomatids. As a first step to understand the importance of Act in these parasites, we have isolated and characterized the act gene of L. major.

EXPERIMENTAL

AND DISCUSSION

(a) Identification of act sequences in L. major by PCR Genomic DNA from L. major promastigotes (LT252 strain, clone CC-l) was purified as previously described (Ozaki and Cseko, 1984). Six degenerate primers targeted to peptide sequences ( 1-DNGSGM, 2-PIEHGIV, 3-MTQIMF, 4-LIGLDEA, 5-SGGTTMF and finally 6-VWIGGSIL) conserved in a wide variety of Act were synthesized. To allow directional cloning, EcoRI and BarnHI linkers were included at the 5’ end of the forward (2 and 3) and reverse (5 and 6) primers, respectively.

124 PCR mixtures

contained

of primers/l.5 mM DNA polymerase/l

50 ng of genomic

DNA/5

FM

MgClJ0.2 mM dNTP/S units Tuq x Tuq polymerase buffer (Promega,

ent gel of intact chromosome DNA containing four different strains of L. mujor and one strain of L. donovuni with the 0.9-kb PCR fragment,

detected

Madison, WI, USA). The initial cycle was performed at 95°C for 4 min, 55°C for 2 min and 72°C for 1 min and

the African

T. brucei, which has a cluster

the subsequent

copies

locates

25 cycles were performed

at 95°C for 1

the region and

of 0.5 Mb (not shown). in chromosomes

a single band at

Thus, in contrast

to

of 2 to 4 gene

of large size class

min, 57°C for 1 min and 72°C for 1 min. An additional

(Ben Amar et al., 1988), L. major act gene has a simpler

10 min at 72°C was added

genomic

amplified

products

to the last cycle. The major

of the expected

size were purified from

agarose gels, digested with EcoRI + BamHI into pBluescript SK(+). Initially, primers

the ends of six clones originated

were sequenced

tical Act encoding generated

pected overlaps

from different

and all of them contained

sequences.

by different

and cloned

Although

sets of primers

arrangement.

The transcription

of uct was confirmed

by Northern

blot analysis of total RNA from promastigotes (CC-l). The 0.9-kb PCR probe recognizes a single transcript of approx.

1.6-kb.

iden-

clones that were exhibited

the ex-

at the 3’ end, none of them displayed

the

forward primer sequences. In addition, all clones had the same 5’ end sequence, suggesting the existence of an internal EcoRI site within the gene. (b) Cloning and characterization of L. major act gene An amplified L. mujor (LT252) genomic library constructed in Xharon 4A (Williams and Blattner, 1979; kindly provided by Dr. S.M. Beverley, Harvard Medical School) was screened using the insert of the longest fully sequenced PCR clone (630 bp). Two clones (Acl and Ac3) were isolated from 2 x 10h pfu. Restriction mapping of both clones showed that they overlap over a region of 7 kb and span a total of 24 kb. The pattern obtained from Southern blots (not shown) containing genomic DNA digested by different restriction enzymes and probed with an 0.9-kb PCR fragment from clone Acl (using as primers 1 and 4) suggests that the act in L. major is a single-copy gene. Three pieces of evidence support this hypothesis. First, the size and number of the BarnHI, BglII, EcoRI and Sac1 fragments from genomic Southern blots are consistent with the restriction map of both clones (not shown). Second, both clones display an identical restriction map over the region in common. Finally, different PCR clones showed the same nt sequence. However, we cannot eliminate the possibility of a second ucr gene, or even a non-functional pseudogene, because two bands are detected in Southern blots of Sal1 and PstI digests. The second band could arise from a second copy, perhaps derived from a duplication of a large region of the chromosome that contains the act gene. A more likely explanation is that there is an allele polymorphism at these sites. Leishmunius are diploid organisms and previous studies have described homologous chromosome polymorphisms (Iovannisci and Beverley, 1989; Blaineau et al., 1991; Curotto de Lafaille and Wirth, 1992). Hybridization of a Southern blot of pulsed-field gradi-

(c) Sequence determination Based on the deduced

and homology with other Act restriction

map of both clones,

we cloned a 0.5-kb XbuI-Eco RI and 1.7-kb Sac1 fragments from clone Acl into pBluescript to determine the entire coding sequence of the act gene. These fragments overlap over 400 bp. Analysis of the sequence reveals one ORF from nt 71 to 1201, which encodes a 42-kDa protein of 377 aa with predicted pl of 5.42 (not shown). When compared with Act sequences from other organisms L. mujor Act is most similar with T. brucei Act (88.6% and 87.2% aa identity, for copies a and b, respectively; Fig. 1). The T. brucei and L. major Act sequences show regions of high divergence (aa positions 2-11, 40-54, 226-240, 261-280 and 309-329) when compared to Act of other organisms (Fig. 1). Analysis of the threedimensional structure of Act show that the diverged regions are dispersed over all four subdomains of monomeric Act (Holmes and Kabsch, 1991). The regions of diverged sequence may explain functional differences described for trypanosomatid Act. For example, H. sumuelpessoui, another trypanosomatid, lacks DNase I binding activity (Mortara, 1989). Immunofluorescence and rhodamine-phalloidin staining of H. sumuelpessoui and T. cruzi revealed that Act is not organized in filaments but display a discrete granular staining pattern. The crystal structure of actin:DNaseI complex and the atomic model of F-Act show that some residues contained in the highly diverged aa may play a major role on actin:DNaseI binding, as well as on contact regions between two monomers of a filament (Holmes et al., 1990; Kabsch et al., 1990). For example, in rabbit skeletal muscle Act, residues Thr”“; G1u2”; Arg3y-Gly4h and Lys61-Ile64 are involved in DNaseI binding (Holmes and Kabsh, 1991; Kabsch and Vandekerckove, 1992). The homologous residues of L. major Act show only 50% of identity (see Fig. 1). The high similarity between L. mujor and T. hrucei Act suggests that Act from other trypanosomatids may also have similar sequences. Thus, it is tempting to speculate that the unique properties of

125 I 1 MADNEQSSIVC

2 3 4 5 6 7 8

1

2 3 4 5 6 7 8

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I

I

I

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130

PblESEtnKAGFSGDDAPRIiVFPSIVGRPKNMQAMMGSANKTVYVGDEAQSKRGVLSL P:EHGIY TNWDDMEKIWHHTFYNDVRVNPEQHNVLLTEAPMNPKQNRE~TFNVPS .S.E..TA...........S.....................E.........KLF......A.....A...............V.......EL.....S...........................G ... .S.E..TA...........S..........L......P...E.......KQEMF......A.....A...............V.......EL .....S......................... ..G..At4 -M.S.VAAL.I......C....A......A.........RHQGI.V.MGQ.DS.......A...I.T.......N...............EL..A..E.P...........S................A F -MEE.IAAL.I......C....A ......A.........RHHGI.V.MGQ.DS.......A...I.T.......................EL..A..E.PC......L...S........I.....A.AF .EGEDVQAL.I......C....A......A.........RHTGV.V.MGQ.DS.......A...I.T.......................EL..A..E.P.......L...A ............... ..T .DED.TTAL.......L............A.........RHQGV.V.MGQ.DS.......A...I.T......I................EL..A..E.PT......L...A .................. -MEE.IAAL........C....A......A.........RHQGV.V.MGQ.DS.......A...I .T..................... ..EL..A..E.P.......L...A ............... ..T I I I 260 I I I I I I I I I LYIGIQLSLYSSGRTTGIVLDAGDGVTHTVPIYEGYSLPHAVRRVDMAGRDLTEYLMKIMMETGTTFTTTAEKERNVKEQLCYVALDFEEEMTNSAKSAN-EEAFELPDGNVMMVGNQRFRCPEVLFKPS .v.......................................I.................L.H..M....S......I...........D.........VS-..P.........Q..........A .. ..A .V.......................................I.................L....M....S.Q....I...........D.........VS-..P.........Q.........QA .. ..A .VS.......A..........S......V....A.F.....IL.I.L......D.....LS.R.YS.S....R..DI..K ....... ..Q..QTA.Q.SSI.KSY.....Q.ITI..E...A..A..H .. .VA.......A..........S..............A....IM.L.L......D.....L..R.Y..S....R..DI..K........ .Q.LQTA.Q.SSL.KSY.....Q.ITI..E...A..A..Q .. .AM.VA....A........M.S....S.........A....IL.L.L......D.....LT.R.YS......R..DI..K.A..... ..Q..QTA.S.SAL.KSY.....Q.ITI..E......A..Q .. .AM.VA....A..........S......N.......A....IM.L.L......D.....LT.R.YS.V....R..DI ..K ....... ..N..ATA.S.SSL.KSY.....Q.ITI..E......T..Q .. .AM.VA....A........M.S..............A....IL.L.L......D.....LT.R.YS......R..DI..K ....... ..Q..ATA.S.SSL.KSY.....Q.ITI..E......A..Q ..

I I I 367 I I I I I I I 1 T.TI;I.nEAPGFPEYQSINKCDIDVRRELYGNIV LSGC.STMFLNLPERLAKEISNLAPSSIKPKVVAPPERKYSYU_G&U,LTTFQTMWVKKSEYDESGPSIVHNKCF 2 ..........H.F............D..........T...K.............P............................ ..S..IT .............S. .. 3 ..........H.F............D..........T...K......G.....................................S..IT.............S ... 4 VL ..- .SA.IDQ.N..M...V...K.......M...T...PGIA..MQ...TA.....M.V.II .....................Q..IS.Q...........H ... 5 AL ..-.NA.IH..N..M...V.I.KD....V.M...T..YPGIAD.MQ...QA.....M.V.I..................S...Q..IS.Q.......G..YR ... 6 FL.M-.SA.IH..N..M...V.I.KD....V.....T...PGIAD.MQ..LTA....TM.I.II.................S...Q..IS.E...........R ...

L.major T.brucef T.brucef(b)

(al

S.cerevfsiae

S.pombe P.polycephalum 7 F..M-.~A.IH..N..M.....I.KD..A.N.M...T..YPGIAD.MQ...TA....TM.I.II.................S...Q..IT.Q....A......R ... Rabbit 8 FL.M-.SC.IH.FN..M...V.I.KD..A.T.....T..YPGIAD.MQ...TA....TM.I.II.................S...Q..IS.Q....A......R ... Human

Fig. 1. Comparison

are indicated. design

the PCR

Schizosucchuromyces

degree degrees

and alignment

The dashes

of identity

represent

of Act aa sequences gaps introduced

from several organisms. to align the sequences.

The residues

The underlined

differing sequences

from their corresponding indicate

the location

aa in L. major Act of the motifs used to

cereuisic~e (70.9%); 5, 1, L. major ; 2, T. brucei, copy a (88.6%); 3, T. brucei, copy b (87.2%); 4, Saccharomycrs 6, Physarum polycephulum (68.9%); 7, rabbit muscle Act (68.8%); 8, human cytoplasmic y-Act (68.8%). The of L. major Act sequence and sequences from other organisms is indicated in between parentheses. The alignment and identity

primers.

pombe (68.5%);

were obtained

by the program MEGALIGN in DNASTAR (Madison, WI, USA). The nt sequence ORF is registered with GenBank (accession No. L16961).

of the genomic

region

encompassing

L.

major act and the translated

the trypanosomatid primary structure.

Act are consistent

with its distinct

(d) Conclusions (I) In spite of the high homology at the protein level between T. brucei and L. major Act sequences, the act gene in these organisms display different genomic organization. The act gene of L. major is probably a singlecopy gene, whereas in T. brucei the act genes are arranged in clusters of 2-4 tandemly linked copies. (2) Although there is a high overall similarity of trypanosomatid Act sequences with Act from higher eukaryotes, some key aa are divergent. This fact might explain the unusual characteristics of the Act of some trypanosomatids.

ACKNOWLEDGEMENTS

We thank Steve Beverley for kindly providing the Kharon 4A genomic library and the CHEF blot and extensive support, Michael Way and Maria C. de Lafaille for critical comments on the manuscript. M.V.de A. was a scholar of the PEW Latin American Program.

REFERENCES Ben Amar, M.F., Pays, A., Tebabi, P., Dero, B., Seebeck, T., Steinart, M. and Pays, E.: Structure and transcription of the actin gene of Trypanosoma hrucei. Mol. Cell. Biol. 8 (1988) 2166-2176. Blaineau. C., Bastien, P., Rioux, J.-A., Roizts, G. and Pages, M.: Long-

range restriction maps of size-variable homologous chromosomes in Leishmania infanturn. Mol. Biochem. Parasitol. 46 ( 1991) 293-302. Curotto de Lafaille, C.A. and Wirth, D.F.: Creation of null/+ mutants of the cc-tubulin gene in Leishmunia enrittii by gene cluster deletion. J. Biol. Chem. 267 (1992) 23839923846. de Souza, W., Meza, I., Martinez-Palomo, A., Souto-Padron, T. and Meirelles, M.N.L.: Trypanosoma cruzi: distribution of fluorescently labeled tubulin and actin in epimastigotes. J. Parasitol. 69 (1983)138~142. Holmes, K.C., Popp, D., Gebhard, W. and Kabsch, W.: Atomic model of the actin filament. Nature 347 (1990) 44449. Holmes, K.C. and Kabsch, W.: Muscle proteins: Actin Curr. Opin. Struct. Biol. 1 (1991) 270-280. Iovannisci. D.M. and Beverley, S.M.: Structural alterations of chromosome 2 in Leishmania major as evidence for diploidy, including spontaneous amplification of the mini-exon array. Mol. Biochem. Parasitol. 34 ( 1989) 1777188. Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F. and Holmes, K.C.: Atomic structure of the actin:DNase I complex. Nature 347 (1990) 37744. Kabsch, W. and Vandekerckove, J.: Structure and function of actin. Annu. Rev. Biophys. Biomol. Struct. 21 (1992) 49976. LeBowitz, J.H., Coburn, C.M., McMahon-Pratt, D., and Beverley, S.M.: Development of a stable Leishumania expression vector and application to the study of parasite surface antigen genes. Proc. Natl. Acad. Sci. USA 87 (1990) 9736-9740. Mortara, R.A.: Studies on trypanosomatid actin. 1. lmmunochemical and biochemical identification. J. Protozool. 36 (1989) 8- 13. Ozaki, L.S. and Czeko, Y.M.T.: Genomic DNA cloning and related techniques. In: Morel, CM. (Ed.), Genes and Antigens of Parasites, A Laboratory Manual. Fundacao Oswald0 Cruz, Rio de Janeiro, 1984, pp. 1655185, Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Williams, B.C. and Blattner, F.R.: Constructuion and characterization of the hybrid bacteriophage lambda Charon vectors for DNA cloning. J. Virol. 29 (1979) 555-575.