Molecular Characterization of a Hyperinducible, Surface Membrane-anchored, Class I Nuclease of a Trypanosomatid Parasite
2000; Elsevier BV; Volume: 275; Issue: 46 Linguagem: Inglês
10.1074/jbc.m004036200
ISSN1083-351X
AutoresMat Yamage, Alain Debrabant, Dennis M. Dwyer,
Tópico(s)HIV/AIDS drug development and treatment
ResumoThe 3′-nucleotidase/nuclease (3′-NT/NU) is a surface enzyme unique to trypanosomatid parasites. These organisms lack the pathway for de novo purine biosynthesis and thus are entirely dependent upon their hosts to supply this nutrient for their survival, growth, and multiplication. The 3′-NT/NU is involved in the salvage of preformed purines via the hydrolysis of either 3′-nucleotides or nucleic acids. In Crithidia luciliae, this enzyme is highly inducible. For example, in these organisms purine starvation triggers an ∼1000-fold up-expression of 3′-NT/NU activity. In the present study, we cloned and characterized a gene encoding this intriguing enzyme from C. luciliae (Cl). Sequence analysis showed that the Cl 3′-NT/NU deduced protein possessed five regions, which we defined here as being characteristic of members of the class I nuclease family. Further, we demonstrated that the Cl 3′-NT/NU-expressed protein possessed both 3′-nucleotidase and nuclease activities. Moreover, we showed that the dramatic up-expression of 3′-NT/NU activity in response to purine starvation of C. luciliae was concomitant with the ∼100-fold elevation in steady-state mRNA specific for this gene. Finally, results of our nuclear run-on analyses demonstrated that such up-regulation in 3′-NT/NU enzyme activity was mediated at the posttranscriptional level. The 3′-nucleotidase/nuclease (3′-NT/NU) is a surface enzyme unique to trypanosomatid parasites. These organisms lack the pathway for de novo purine biosynthesis and thus are entirely dependent upon their hosts to supply this nutrient for their survival, growth, and multiplication. The 3′-NT/NU is involved in the salvage of preformed purines via the hydrolysis of either 3′-nucleotides or nucleic acids. In Crithidia luciliae, this enzyme is highly inducible. For example, in these organisms purine starvation triggers an ∼1000-fold up-expression of 3′-NT/NU activity. In the present study, we cloned and characterized a gene encoding this intriguing enzyme from C. luciliae (Cl). Sequence analysis showed that the Cl 3′-NT/NU deduced protein possessed five regions, which we defined here as being characteristic of members of the class I nuclease family. Further, we demonstrated that the Cl 3′-NT/NU-expressed protein possessed both 3′-nucleotidase and nuclease activities. Moreover, we showed that the dramatic up-expression of 3′-NT/NU activity in response to purine starvation of C. luciliae was concomitant with the ∼100-fold elevation in steady-state mRNA specific for this gene. Finally, results of our nuclear run-on analyses demonstrated that such up-regulation in 3′-NT/NU enzyme activity was mediated at the posttranscriptional level. nucleotidase/nuclease amino acid(s) polyacrylamide gel electrophoresis base pair(s) polymerase chain reaction pyrimidine purine open reading frame pulsed field gel electrophoresis untranslated region nucleotide(s) kilobase pair(s) rapid amplification of cDNA ends digoxigenin Trypanosomatid protozoa are a group of primitive parasitic organisms, which are of importance because many of them cause serious or fatal diseases in humans and domestic animals worldwide (54UNDP/World Bank/WHO Tropical Research: Progress 1995–96; Thirteenth Programme Report of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. World Health Organization, Geneva, Switzerland1997Google Scholar). Although purines are essential for the growth, multiplication and survival of these organisms, they are incapable of synthesizing the purine ring de novo. Thus, they are totally dependent upon their hosts to provide an exogenous source of preformed purines (1Bryant C. Behm C.A. Biochemical Adaptation in Parasites. Chapman and Hall, New York.1989: 71-91Google Scholar). In that regard, these purine auxotrophic organisms possess extracellular salvage mechanisms to acquire these essential nutrients from their host environments (2Hammond D.J. Gutteridge W.E. Mol. Biochem. Parasit. 1984; 13: 243-261Crossref PubMed Scopus (223) Google Scholar). One such trypanosomatid salvage enzyme is the externally oriented, bifunctional, surface membrane 3′-nucleotidase/nuclease (3′-NT/NU),1 which is capable of generating free nucleosides via the hydrolysis of either 3′-nucleotides or nucleic acids (3Gottlieb M. Parasitol. Today. 1989; 5: 257-260Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 4Bates P.A. Coombs G.H. North M.J. Biochemical Protozoology. Taylor & Francis, Washington, D. C.1991: 537-553Google Scholar). Such 3′-NT/NU activities have been reported from a variety of different trypanosomatids (4Bates P.A. Coombs G.H. North M.J. Biochemical Protozoology. Taylor & Francis, Washington, D. C.1991: 537-553Google Scholar, 5Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (69) Google Scholar). Among these organisms, however, only the 3′-NT/NU of Crithidia luciliae was shown to be dramatically up-expressed by ∼1000-fold in response to purine starvation conditions (6Gottlieb M. Science. 1985; 227: 72-74Crossref PubMed Scopus (53) Google Scholar). Further, the C. luciliae 3′-NT/NU was shown to possess some biochemical and kinetic properties similar to those of the class I, single-strand-specific, nucleases of fungi and germinating plant seedlings (3Gottlieb M. Parasitol. Today. 1989; 5: 257-260Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 7Neubert T.A. Gottlieb M. J. Biol. Chem. 1990; 265: 7236-7242Abstract Full Text PDF PubMed Google Scholar). In light of these intriguing biochemical and physiological properties, experiments were designed in the current report to delineate the structure, function, and expression of the gene encoding this highly inducible enzyme of C. luciliae. Reagents used in this study were purchased from Sigma unless otherwise specified. Cultures of C. luciliae (ATCC strain no. 30285) (8Alleman M.M. Mann V.H. Bacchi C.J. Yarlett N. Gottlieb M. Dwyer D.M. Exp. Parasitol. 1995; 81: 519-528Crossref PubMed Scopus (11) Google Scholar) were grown at 26 °C in medium M199 (Life Technologies, Inc.), pH 6.8, as described previously (9McCarthy-Burke C. Bates P. Dwyer D.M. Exp. Parasitol. 1991; 73: 385-387Crossref PubMed Scopus (49) Google Scholar) and supplemented here with 10% (v/v) heat-inactivated fetal bovine serum (Life Technologies, Inc.). A clone (MY101) was isolated by limiting dilution from such cultures and used for all subsequent experiments. For testing the effects of adenosine depletion on C. luciliae, replete cells were grown and maintained in chemically defined medium, RPMI 1640+ (9McCarthy-Burke C. Bates P. Dwyer D.M. Exp. Parasitol. 1991; 73: 385-387Crossref PubMed Scopus (49) Google Scholar) with 100 μm adenosine, and "adenosine-starved" cells were incubated in such medium without adenosine for various periods up to 72 h. In all experiments, log-phase cell cultures (2–4 × 107/ml) were harvested by centrifugation as described previously (10Bates P.A. Hermes I. Dwyer D.M. Mol. Biochem. Parasitol. 1990; 39: 247-256Crossref PubMed Scopus (80) Google Scholar). Both the cells and the resulting cell-free culture supernatants were processed for various purposes as described below. For some experiments, C. luciliae were treated with 0.5 μg/ml tunicamycin as described previously (11Bates P.A. Dwyer D.M. Mol. Biochem. Parasitol. 1987; 26: 289-296Crossref PubMed Scopus (62) Google Scholar, 12Doyle P.S. Dwyer D.M. Exp. Parasitol. 1993; 77: 435-444Crossref PubMed Scopus (28) Google Scholar). The designations used in this report for genes, proteins, and plasmids follow the genetic nomenclature forTrypanosoma and Leishmania as outlined by Claytonet al. (13Clayton C. Adams M. Almeida R. Baltz T. Barret M. Bastien P. Belli S. Beverley S. Biteau N. Blackwell J. Blaineau C. Boshart M. Bringaud F. Cross G. Cruz G. Cruz A. Degrave W. Donelson J. El-Sayed N. Fu G. Ersfeld K. Gibson W. Gull K. Ivens A. Kelly J. Lawson D. Lebowitz J. Majiwa P. Mathews K. Melville S. Merlin G. Michels P. Myler P. Norrish A. Opperdoes F. Papadopoulou B. Parsons M. Seebeck T. Smith D. Stuart K. Turner M. Ullu E. Vanhamme L. Mol. Biochem. Parasitol. 1998; 97: 221-224Crossref PubMed Scopus (79) Google Scholar). Degenerate oligonucleotide primers were designed based on the amino acid sequences conserved among the Leishmania donovani 3′-nucleotidase/nuclease (5Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (69) Google Scholar), Aspergillus oryzae S1 nuclease (14Iwamatsu A. Aoyama H. Dibo G. Tsunasawa S. Sakiyama F. J. Biochem. (Tokyo). 1991; 110: 151-158Crossref PubMed Scopus (38) Google Scholar), and Penicillium citrinum P1 nuclease (15Maekawa K. Tsunasawa S. Dibo G. Sakiyama F. Eur. J. Biochem. 1991; 200: 651-661Crossref PubMed Scopus (49) Google Scholar) following a codon usage of C. luciliae RNA polymerase II (16Croan D.G. Ellis J. Mol. Biochem. Parasitol. 1996; 79: 97-102Crossref PubMed Scopus (33) Google Scholar). Such primers were synthesized by β-cyanoethylphosphoramidite chemistry using an Expedite nucleic acid synthesis system (PE Applied Biosystems, Foster City, CA). Primer 1 (P1) (forward, 5′-GG(T CG)G A(CT) AT(C T)CA (AG)C CIC T(GC T)CA-3′) and primer 2 (P2) (reverse, 5′-(TC)T T(CG A)GC IAG ICG GTA (GCA) CC-3′) were used in PCR amplification of C. luciliae cDNA withTaq polymerase (Roche Molecular Biochemicals). Conditions for amplification were 94 °C for 30 s, 53 °C for 1 min, 72 °C for 2 min (36 cycles), and 74 °C for 10 min. The 436-nt product of this reaction was ligated to pCRII by TA cloning (Invitrogen, Carlsbad, CA), and the resulting plasmid was designatedpCl-436. Both strands of this plasmid were sequenced and found to have high homology with that of the L. donovani3′-NT/NU gene (5Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (69) Google Scholar). The pCl-436 insert was labeled with digoxigenin-dUTP (Roche Molecular Biochemicals) by PCR according to manufacturer's instructions. The resulting digoxigenin-labeled probe (DIG-436) was used for screening the C. luciliae cosmid library and for other hybridization studies. Genomic DNA was isolated from 109 washed C. luciliae using the GNome DNA isolation kit (Bio101, Vista, CA). A C. luciliaegenomic DNA library was constructed from EcoRV (Roche Molecular Biochemicals)-restricted genomic DNA, blunt end-ligated to a SuperCos I vector, phage-packaged and adsorbed onto hostEscherichia coli XL1 Blue MR all according to manufacturer's instructions (Stratagene, La Jolla, CA). Over 3000 colonies from this library were screened by hybridization with the DIG-436 probe at high stringency (hybridization and washing in 0.1× SSC and 0.1% SDS at 65 °C). Several positive clones were obtained. A 2.8-kb NcoI fragment from one of these cosmid clones (CosMY1) was subcloned into PCRScript (Stratagene), and the resulting plasmid (pCl-3) was used for sequence analysis. Plasmid DNA was sequenced using the fluorescent dideoxy-chain terminator method of cycle sequencing (Dye Terminator Cycle Sequencing Ready Reaction kit, PerkinElmer Life Sciences) on a model 373A Applied Biosystems automated DNA sequencer. Sequence data from both strands were analyzed using the Wisconsin Package, version 10.0, Genetic Computer Group (GCG) software package running on the National Institutes of Health Unix System and Sequencher 3.0 software (Gene Codes Corp., Ann Arbor, MI). Protein multiple sequence alignments were done using the ClustalW program (17Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56657) Google Scholar). For Southern blotting, C. luciliaegDNA was digested with various restriction endonucleases (AccI, AvaI, BglI, andSalI, Roche Molecular Biochemicals), separated by 1% agarose gel electrophoresis, transferred under vacuum onto nylon membranes (HyBond-N, Amersham Pharmacia Biotech) and cross-linked by UV irradiation. DIG-labeled PCR products were generated using thepCl-3 insert above as template and the appropriate oligonucleotide primers specific to the 5′-flanking (DIG-300, which spans nt −980 to nt −664) and 3′-flanking (DIG-420, which spans nt +864 to nt +1291) regions of the Cl 3′-NT/NU gene, respectively. Membranes were processed for hybridization at high stringency with either the DIG-300- or DIG-420-labeled probes according to the Genius system users' guide (Roche Molecular Biochemicals), and the hybridized fragments were visualized using the Genius detection system (Roche Molecular Biochemicals). Agarose plugs containing ∼1 × 108 washed C. luciliae or C. fasciculata were prepared for PFGE according to previously described methods (18Pogue G.P. Joshi M. Lee N.S. Dwyer D.M. Kenny R.T. Gam A.A. Nakhasi H.L. Mol. Biochem. Parasitol. 1996; 81: 27-40Crossref PubMed Scopus (17) Google Scholar). Chromosomes were separated by PFGE in a Bio-Rad DRII PFGE apparatus using conditions described by Joshi et al. (19Joshi M. Dwyer D.M. Nakhasi H.L. Mol. Biochem. Parasitol. 1993; 58: 345-354Crossref PubMed Scopus (97) Google Scholar). Gels were stained with ethidium bromide, blotted onto nylon membranes (Hybond-N, Amersham Pharmacia Biotech), and processed for Southern blot hybridization with the DIG-436 probe as above. cDNA analyses were done in order to characterize the 5′-splice acceptor site and 3′-polyadenylation site of the Cl 3′-NT/NU gene. Since C. luciliae up-regulate their expression of 3′-nucleotidase activity in response to purine starvation (6Gottlieb M. Science. 1985; 227: 72-74Crossref PubMed Scopus (53) Google Scholar), mRNA was isolated from such cells using Poly(A) Pure (Ambion, Austin, TX). cDNA was synthesized by reverse transcription of poly(A)+ RNA obtained from cells starved for adenosine for 24 h using SuperScript II (Life Technologies, Inc.) and an oligo(dT)14 adaptor (5′-AGA AGA CGT AGG TTG ACT GCT GCA GTT TTT TTT TTT TTT-3′). Following RNase H (Roche Molecular Biochemicals) treatment, the resulting cDNA was used as template for PCR. In such reactions, the 5′ and 3′ ends of the Cl 3′-NT/NU cDNA were generated using the RACE method (20Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4491) Google Scholar). For 5′-RACE (i.e.splice acceptor site), forward primer was 5′-AAC GCT ATA TAT AAG TAT CAG TTT C-3′ and reverse was 5′-GAG AAC TCG TTG GCG TTG T-3′. For 3′-RACE (i.e. polyadenylation site), forward primer was 5′-ACC TAC GCC TCT ACG CTG A-3′ and adaptor was 5′-AGA AGA CGT AGG TTG ACT G-3′. PCRs were carried out using conditions as above, and the resulting fragments were also cloned into pCRII (Invitrogen) as above. Subsequent sequencing of these fragments verified them as a part of theC. luciliae 3′-nucleotidase/nuclease gene. Full-length Cl 3′-NT/NU cDNA was generated by XL-PCR (Roche Molecular Biochemicals) using the 5′-RACE forward and 3′-RACE adapter primers and the cDNA synthesized above. The resulting 2.2-kb PCR product was ligated into PCRScript (Stratagene). Subsequently, this cloned insert was sequenced. Those results showed that this cDNA clone possessed sequences identical to those obtained from the 5′- and 3′-RACE products, as well as the Cl 3′-NT/NU ORF present in the pCl-3 genomic clone, above. A truncated construct of the Cl 3′-NT/NU gene was generated by PCR using pCl-3 as template and the appropriate oligonucleotide primers. The resulting fragment contained the Cl 3′-NT/NU 5′-UTR (nt −105 to nt −1) and a portion of the Cl 3′-NT/NU ORF (nt +1 to nt +1005). The resulting ∼1.1-kb PCR fragment was digested withSpeI and ligated into the SpeI site of the [pKSNEO] leishmanial expression vector (21Zhang W.W. Charest H. Ghedin E. Matlashewski G. Mol. Biochem. Parasitol. 1996; 78: 79-90Crossref PubMed Scopus (122) Google Scholar). The resulting plasmid [pKSNEO-Cl 3′-nt/nu ΔC] and the [pKSNEO] control plasmid were transfected intoC. luciliae cells by electroporation essentially as described by Descoteaux et al. (22Descoteaux A. Garraway L.A. Ryan K.A. Garrity L.K. Turco S.J. Beverley S.M. Methods Mol. Genet. 1994; 3: 22-48Google Scholar). Following an overnight recovery in medium M199+ (9McCarthy-Burke C. Bates P. Dwyer D.M. Exp. Parasitol. 1991; 73: 385-387Crossref PubMed Scopus (49) Google Scholar) with 10% fetal bovine serum at 26 °C, these cells were selected for their growth under increasing concentrations of Geneticin (G-418, Life Technologies, Inc.) up to 200 μg/ml. In some experiments, C. luciliae were treated with 0.5 μg/ml tunicamycin as described previously (11Bates P.A. Dwyer D.M. Mol. Biochem. Parasitol. 1987; 26: 289-296Crossref PubMed Scopus (62) Google Scholar, 12Doyle P.S. Dwyer D.M. Exp. Parasitol. 1993; 77: 435-444Crossref PubMed Scopus (28) Google Scholar). Lysates and cell-free culture supernatants from these transfectants were used in various experiments. 3′-Nucleotidase enzyme activity was measured in cell lysates and in cell-free culture supernatants in test tube assays, using adenosine 3′-monophosphate (3′-AMP) as substrate, as described previously (7Neubert T.A. Gottlieb M. J. Biol. Chem. 1990; 265: 7236-7242Abstract Full Text PDF PubMed Google Scholar). One unit of enzyme activity is defined as 1 nmol of Pi released from the 3′-AMP/min per ml of cell-free culture supernatant or per mg of cell lysate protein. Protein concentrations were determined using the bicinchoninic acid method (BCA, Pierce). Similar samples of C. luciliae were also assayed for total nuclease activity using poly(A) as substrate as described previously (23Gottlieb M. MacKow M.C. Neubert T.A. Exp. Parasitol. 1988; 66: 108-117Crossref PubMed Scopus (21) Google Scholar). Cell-free culture supernatants and cell lysates ofC. luciliae were separated by SDS-PAGE. These gels were processed for either in situ staining for 3′-nucleotidase activity (24Zlotonick G.W. Mackow M.C. Gottlieb M. Comp. Biochem. Physiol. 1987; 87B: 629-635Google Scholar, 7Neubert T.A. Gottlieb M. J. Biol. Chem. 1990; 265: 7236-7242Abstract Full Text PDF PubMed Google Scholar) or in situ staining for nuclease activity (25Bates P.A. FEMS Microbiol. Lett. 1993; 10: 53-58Crossref Scopus (35) Google Scholar). Similar SDS-PAGE gels were processed for Western blot analysis with various rabbit antisera or their preimmune sera as described previously (5Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (69) Google Scholar). One of these was a rabbit antiserum (no. 1336, anti-Ld3′-NT/NU-specific) generated against a single internal peptide of the L. donovani 3′-nucleotidase/nuclease (amino acid residues Glu201–Tyr226) (5Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (69) Google Scholar). A second antiserum (no. 1398) was generated in a New Zealand White rabbit against an E. coli-expressed Ld 3′-NT/NU protein, which lacked both the N-terminal 25-aa signal peptide and the C-terminal 44 aa residues of the Ld 3′-NT/NU. Preimmune sera from these rabbits were used as controls. A DIG-labeled 1000-nt PCR product (DIG-1000), which spans nt 1–1005 of the Cl 3′-NT/NU ORF, was generated using the pCl-3 insert above as template and the appropriate oligonucleotide primers. An identical32P-labeled probe (32P-1000) was similarly generated. These probes were used in Northern blot analyses to quantitate the steady-state levels of specific mRNA present in adenosine-replete and adenosine-starved cells. Total RNAs were isolated from both adenosine-replete and adenosine-starved cells using STAT60 (Tel-Test, Inc., Friendswood, TX). Equivalent amounts (20 μg) of these RNAs were separated by agarose gel electrophoresis (26Charest H. Matlashewski G. Mol. Cell. Biol. 1994; 14: 2975-2984Crossref PubMed Scopus (140) Google Scholar), transferred onto nylon membranes (HyBond-N, Amersham Pharmacia Biotech), and cross-linked by UV irradiation. Blots were hybridized at high stringency with either the DIG-1000 probe using Genius detection system (Roche Molecular Biochemicals) as above or with the32P-1000 probe as described previously (26Charest H. Matlashewski G. Mol. Cell. Biol. 1994; 14: 2975-2984Crossref PubMed Scopus (140) Google Scholar). Images obtained with the DIG-labeled probe were analyzed using the National Institutes of Health Image densitometry software package (available via the National Institutes of Health web site) and those obtained with the 32P-1000 labeled probe were quantified by phosphorimaging analyses using a PhosphorImager (model SI, Molecular Dynamics, Sunnyvale, CA) driven by a Scanner Softwaremanager package (Molecular Dynamics). The above blots were subsequently rehybridized with either a 440-nt DIG- or 32P-labeled fragment of a cloned constitutively expressed C. luciliae α-tubulin gene ORF. 2M. Yamage, unpublished data. Nuclear run-on experiments were done with adenosine-replete and adenosine-starved cells to ascertain the rates at which they transcribed the Cl 3′-NT/NU gene. Nuclei were isolated from C. luciliae maintained for 24 h in either adenosine-replete or adenosine-starved medium using previously described methods (27Mahmood R. Ray D.S. J. Biol. Chem. 1998; 273: 23729-23734Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Run-on transcriptions with [α-32P]rUTP (Amersham Pharmacia Biotech) were performed as described previously (28Burchmore R.J.S. Landfear S.M. J. Biol. Chem. 1998; 273: 29118-29126Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and similar experiments were also done using digoxigenin-UTP (Roche Molecular Biochemicals). Slot blots were prepared on HyBond-N (Amersham Pharmacia Biotech) membranes containing equivalent amounts of either a 436-bp internal DNA fragment (nt +446 to nt +881) of the Cl 3′-NT/NU gene, a similar sized (440 bp) internal fragment of the C. luciliae α-tubulin gene above, or a control linearized pBluescript plasmid (Stratagene). Such blots were hybridized with either the 32P-labeled or DIG-labeled nascent mRNAs, and the resulting signals were captured and analyzed as above. A 436-bp DNA fragment was obtained by RT-PCR using cDNA (derived from cells starved for adenosine for 12 h) as a template and a pair of the degenerate oligonucleotide primers: P1 and P2 (see "Experimental Procedures"). This fragment was cloned, sequenced, DIG-labeled, and used as a probe (DIG-436) to screen a C. luciliae gDNA cosmid library. Of the several clones identified from such screening, one cosmid clone (Cos MY1) was chosen for further analysis. Following restriction digestion of Cos MY1, a 2.8-kb NcoI fragment was isolated and subcloned into PCRScript and the resulting plasmid, pCl-3, was used for sequence analysis. Both strands of the cloned NcoI fragment were sequenced. Results of sequence analysis showed that the pCl-3 plasmid contained a complete ORF of 1,134 bp (Cl 3′-NT/NU, Fig.1). This ORF showed high nt sequence identity (69%) to the L. donovani 3′-nucleotidase/nuclease gene (5Debrabant A. Gottlieb M. Dwyer D.M. Mol. Biochem. Parasitol. 1995; 71: 51-63Crossref PubMed Scopus (69) Google Scholar). Moreover, sequence analysis revealed that G or C was the preferred base used at the third codon position in the Cl 3′-NT/NU. Out of 378 codons including TAG stop in this ORF, nearly 90% contained a C or G at the third nucleotide position (186 end with C and 154 end with G). Further, the overall composition of the Cl 3′-NT/NU is very GC-rich (65.0%).Figure 1Nucleotide and deduced amino acid sequences of the C. luciliae 3′-nucleotidase/nuclease (Cl 3′-NT/NU). Nucleotide numbers are shown at theleft; those constituting the 5′-UTR and 5′-flanking region of this gene are designated (−). The ORF (nt+1 to nt+1134) is shown inuppercase letters, and the 5′- and 3′-flanking regions are shown in lowercase letters. The TAG stop codon of the ORF is denoted AMB. The P1 and P2arrows designate the oligonucleotide primers used to generate the 436-bp PCR amplification product. The putative splice acceptor site ag (nt −106/−107) in the 5′-UTR and the putative polyadenylation aataaa motifs in the 3′-UTR are shown in bold type. Putative CAAT and TATA boxes are underlined in the 5′-flanking region. The deduced amino acid sequence is shown initalics, and residues are numbered on theright. The 25-aa predicted signal peptide sequence is denoted with a dashed underline. The 25-aa putative trans-membrane-spanning anchor region (Gly336–Leu360) is underlined inbold. The C-terminal 17-aa residues constitute a putative cytoplasmic tail. Asterisks indicate the two predictedN-glycosylation sites (Asn240 and Asn294).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The relationship between the pCl-3 gDNA clone, a full-length cDNA Cl 3′-NT/NU clone and four DIG-labeled probes (i.e. DIG-300, -420, -436, and -1000) generated from these clones is shown diagrammatically in Fig.2 A. The combined results of restriction digestion of these two clones are summarized as a restriction map in Fig. 2 B. It is of importance to note that, with only a few exceptions (29Schneider A. McNally K.P. Agabien N. J. Biol. Chem. 1993; 268: 21868-21874Abstract Full Text PDF PubMed Google Scholar), trypanosomatid protozoa generally do not possess introns within their ORFs (30Fong D. Lee B. Mol. Biochem. Parasitol. 1988; 31: 97-106Crossref PubMed Scopus (45) Google Scholar). Further, all pre-mRNAs in these organisms are joined to a 39-nt conserved spliced leader at their 5′ end by trans-splicing (31Borst P. Annu. Rev. Biochem. 1986; 55: 701-732Crossref PubMed Scopus (304) Google Scholar, 32Agabian N. Cell. 1990; 61: 1157-1160Abstract Full Text PDF PubMed Scopus (299) Google Scholar). To further characterize the organization of the Cl 3′-NT/NU, we compared the sequence of our genomic pCl-3 clone with that of the full-length cDNA clone. Results of these analyses revealed that the first in-frame start codon, ATG, was preceded by 105 bp of 5′-UTR (Figs. 1 and 2 B). The PyAG spliced-leader acceptor site was mapped to −106 bp from the start codon (Figs. 1 and2 B). As shown in Fig. 1, regions upstream of this site contained the sequences: CCTTGAC (−220 to −214), TGTTGAC (−156 to −150), and TTTCAAC (−125 to −119), which conform to the PyNPyPyPuAPy consensus (33Lewin B. Genes VII. Oxford University Press, Oxford, United Kingdom2000Google Scholar). In the latter, nt A is the potential target for lariat formation during the pre-mRNA trans-splicing events (34Reed R. Maniatis T.R. Cell. 1985; 41: 95-105Abstract Full Text PDF PubMed Scopus (265) Google Scholar). Further, short segments typical of canonical CAAT and TATA boxes, which are present in higher eukaryotes, but remain to be defined in trypanosomatid protozoa, were also found in the 5′-UTR of theCl 3′-NT/NU gene at −421 and −384 bp, respectively (Fig.1). Similar analyses of the region downstream from the 3′ end of theCl 3′-NT/NU ORF revealed three putative polyadenylation signals (AATAAA) at nt 2,062, 2,131, and 2,135 (Figs. 1 and2 B). These signals could direct a poly(A) tail to be added downstream of one of these sites. To assess the structure and copy number of theCl 3′-NT/NU, genomic DNA was digested with several restriction endonucleases and subjected to Southern hybridization with the DIG-labeled probes shown in Fig. 2 A. These enzymes were chosen because they possessed at least one predicted restriction site within the Cl 3′-NT/NU ORF (Fig. 2 B). Results of Southern hybridizations obtained with the DIG-300 probe (i.e. the 5′-flanking region of pCl-3) and with the DIG-420 probe (i.e. the 3′-flanking region ofpCl-3) are shown in Fig. 2 (C and D), respectively. The cumulative results of these analyses indicated that at least three copies of the Cl 3′-NT/NU gene are present within the diploid genome of C. luciliae. To identify the physical location(s) of the Cl 3′-NT/NUgenes, chromosomal DNA from C. luciliae and C. fasciculatawas separated by pulsed-field gel electrophoresis and stained with ethidium bromide (Fig. 2 E, lanes 1 and2). These gels were blotted onto nylon membranes and hybridized with the Cl 3′-NT/NU DIG-436 probe above. Results of these assays demonstrated that the Cl 3′-NT/NU genes were all apparently localized to a single >1.6-megabase chromosome inC. luciliae (Fig. 2 E, lane 3) and to a similarly sized chromosome in Crithidia fasciculata (Fig. 2 E, lane 4). The latter is in agreement with a previous observations which demonstrated that C. fasciculata possess 3′-nucleotidase and nuclease activities (35Hassan H.F. Coombs G.H. Mol. Biochem. Parasitol. 1987; 23: 285-296Crossref PubMed Scopus (49) Google Scholar). Taken together, these results indicate that the 3′-NT/NU gene is conserved among these closely related species of trypanosomatid parasites. TheCl 3′-NT/NU open reading frame of 1,134 bp encodes a deduced protein of 377 amino acids with a calculated mass of 40,575 Da (Fig.1). This deduced protein is acidic (∼16% acidic aa residues) and has a predicted isoelectric point (pI) of 5.63, which is in agreement with the pI of ∼5.8 as determined previously by two-dimensional electrophoresis of the native C. luciliae enzyme (7Neubert T.A. Gottlieb M. J. Biol. Chem. 1990; 265: 7236-7242Abstract Full Text PDF PubMed Google Scholar). Hydropathy analysis using the Kyte and Doolittle algorithm (46Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17916) Google Scholar) revealed that the Cl 3′-NT/NU deduced protein possessed a hydrophobic domain at its N terminus (Fig. 2 G). Based on the von Heijne algorithm (36Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4970) Google Scholar
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