Isolation of Trypanosoma brucei CYC2 andCYC3 Cyclin Genes by Rescue of a Yeast G1Cyclin Mutant
2000; Elsevier BV; Volume: 275; Issue: 12 Linguagem: Inglês
10.1074/jbc.275.12.8315
ISSN1083-351X
AutoresJaap J. van Hellemond, Philippe Neuville, Ralph Τ. Schwarz, Keith R. Matthews, Jeremy C. Mottram,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoTwo Trypanosoma brucei cyclin genes,CYC2 and CYC3, have been isolated by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G1 cyclin function. CYC2 encodes a 24-kDa protein that has sequence identity to the Neurospora crassa PREG1 and the S. cerevisiae PHO80 cyclin. CYC3 has the most sequence identity to mitotic B-type cyclins from a variety of organisms. Both CYC2 and CYC3 are single-copy genes and expressed in all life cycle stages of the parasite. To determine if CYC2 is found in a complex with previously identified trypanosome cdc2-related kinases (CRKs), theCYC2 gene was fused to the TY epitope tag, integrated into the trypanosome genome, and expressed under inducible control. CYC2ty was found to associate with an active trypanosome CRK complex since CYC2ty bound to leishmanial p12cks1, and histone H1 kinase activity was detected in CYC2ty immune-precipitated fractions. Gene knockout experiments provide evidence thatCYC2 is an essential gene, and co-immune precipitations together with a two-hybrid interaction assay demonstrated that CYC2 interacts with CRK3. The CRK3·CYC2ty complex, the first cyclin-dependent kinase complex identified in trypanosomes, was localized by immune fluorescence to the cytoplasm throughout the cell cycle. Two Trypanosoma brucei cyclin genes,CYC2 and CYC3, have been isolated by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G1 cyclin function. CYC2 encodes a 24-kDa protein that has sequence identity to the Neurospora crassa PREG1 and the S. cerevisiae PHO80 cyclin. CYC3 has the most sequence identity to mitotic B-type cyclins from a variety of organisms. Both CYC2 and CYC3 are single-copy genes and expressed in all life cycle stages of the parasite. To determine if CYC2 is found in a complex with previously identified trypanosome cdc2-related kinases (CRKs), theCYC2 gene was fused to the TY epitope tag, integrated into the trypanosome genome, and expressed under inducible control. CYC2ty was found to associate with an active trypanosome CRK complex since CYC2ty bound to leishmanial p12cks1, and histone H1 kinase activity was detected in CYC2ty immune-precipitated fractions. Gene knockout experiments provide evidence thatCYC2 is an essential gene, and co-immune precipitations together with a two-hybrid interaction assay demonstrated that CYC2 interacts with CRK3. The CRK3·CYC2ty complex, the first cyclin-dependent kinase complex identified in trypanosomes, was localized by immune fluorescence to the cytoplasm throughout the cell cycle. The parasitic protozoon Trypanosoma brucei belongs to the family of Trypanosomatidae, which is thought to be one of the earliest branching eukaryotic families to contain a mitochondrion (1.Sogin M.L. Curr. Opin. Genet. Dev. 1991; 1: 457-463Crossref PubMed Scopus (397) Google Scholar). The African trypanosome, T. brucei, is responsible for the clinically important diseases, sleeping sickness in humans and nagana in cattle. Trypanosomes have a complex life cycle that alternates between insect and mammalian hosts with different developmental forms having specific roles within each host. The rapidly dividing forms such as the long slender bloodstream form in the mammal or the procyclic form in the gut of the tsetse fly vector establish an infection in the host. The parasite also has a nondividing short stumpy bloodstream form, which is arrested in the G0/G1 phase of the cell cycle and is preadapted for survival when the organism passes into the tsetse (2.Vickerman K. Br. Med. Bull. 1985; 41: 105-114Crossref PubMed Scopus (478) Google Scholar). During the trypanosome life cycle there is an integral link between the control of the cell cycle and parasite differentiation (3.Matthews K.R. Gull K. Parasitol. Today. 1994; 10: 473-476Abstract Full Text PDF PubMed Scopus (40) Google Scholar), and there is a special need to coordinate replication and segregation of its single organelles: the nucleus, mitochondrion, and flagellum (4.Woodward R. Gull K. J. Cell Sci. 1990; 95: 49-57Crossref PubMed Google Scholar). Thus, trypanosomes have added complexity in cell cycle control not present in many single-cell eukaryotes such as yeast. The similarities and differences between the regulation of the cell cycle in trypanosomatids and other eukaryotes provide important information about the evolution of regulatory systems controlling the cell cycle. However, little is known in molecular terms of how the trypanosome cell cycle is controlled. Cell cycle progression in eukaryotes is regulated by a family of cyclin-dependent kinases (CDKs) 1The abbreviations used are; CDK, cyclin-dependent kinase; CRK, cdc2-related kinase; MOPS, 4-morpholinepropanesulfonic acid; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); PARP, procyclic acidic repetitive protein. that require association with cyclin regulatory partners for activity. The first CDKs identified, Schizosaccharomyces pombe cdc2 andSaccharomyces cerevisiae CDC28, are highly conserved in evolution and were the first of the CDK family (CDK1) to be shown to have a central role in cell cycle control (5.Nurse P. Nature. 1990; 344: 503-508Crossref PubMed Scopus (2225) Google Scholar, 6.Nasmyth K. Curr. Opin. Cell Biol. 1993; 5: 166-179Crossref PubMed Scopus (409) Google Scholar). CDKs have since been shown to have roles in other cellular functions such as transcriptional regulation and response to stress (7.Hardie D.G. Cell. Signal. 1994; 6: 813-821Crossref PubMed Scopus (22) Google Scholar). The activity of CDKs can be controlled post-translationally by a number of different mechanisms: the association of the kinase subunit with its positive regulatory cyclin partner, phosphorylation of conserved sites, which can stimulate or inhibit kinase activity, and the binding of CDK inhibitor proteins (8.Johnson D.G. Walker C.L. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 295-312Crossref PubMed Scopus (567) Google Scholar). Since binding of a cyclin partner is essential for full CDK activity, the concentrations of the cyclins are tightly controlled by transcriptional regulation as well as a timing of proteolysis (9.Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (498) Google Scholar). In addition to the concentration of the cyclin “cycling” during the cell cycle, cyclins are characterized by the presence of a 100-amino acid region of sequence similarity to the consensus cyclin box, which is involved in binding to their CDK partner (10.Brown N.R. Noble M.E.M. Endicott J.A. Garman E.F. Wakatsuki S. Mitchell E. Rasmussen B. Hunt T. Johnson L.N. Structure. 1995; 3: 1235-1247Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 11.Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massagué J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1216) Google Scholar). So far, a large family of cyclin genes has been identified in many species, of which the majority are involved in regulation of the cell cycle (8.Johnson D.G. Walker C.L. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 295-312Crossref PubMed Scopus (567) Google Scholar). Mitotic cyclins contain a characteristic nine-amino acid destruction box motif, which targets cyclins for degradation at the end of mitosis (12.Glotzer M. Murray A.W. Kirschner M. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1901) Google Scholar). G1 cyclins are more divergent, and some have C-terminal PEST sequences, which are involved in their degradation (13.Arnaud L. Pines J. Nigg E.A. Chromosoma (Berl.). 1998; 107: 424-429Crossref PubMed Scopus (119) Google Scholar). The budding yeast CDKs, CDC28 and PHO85, bind at least three different classes of cyclin, each containing between two and five members, giving a high level of functional redundancy (14.Andrews B. Measday V. Trends Genet. 1998; 14: 66-72Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Higher eukaryotes, on the other hand, have a multiplicity of CDKs (nine at the last count) and cyclins (A-T) (8.Johnson D.G. Walker C.L. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 295-312Crossref PubMed Scopus (567) Google Scholar). A complex regulation of the cell cycle in trypanosomatids is supported by the identification of a large family of genes with homology to CDKs. These genes were termed cdc2-relatedkinase (CRK) genes, as no cyclin partners were known to bind these kinases when they were first described. CRKs have been identified in T. brucei (15.Mottram J.C. Smith G. Gene. 1995; 162: 147-152Crossref PubMed Scopus (64) Google Scholar, 16.Mottram J.C. Parasitol. Today. 1994; 10: 253-257Abstract Full Text PDF PubMed Scopus (44) Google Scholar), Leishmania(17.Mottram J.C. Kinnaird J. Shiels B.R. Tait A. Barry J.D. J. Biol. Chem. 1993; 268: 21044-21051Abstract Full Text PDF PubMed Google Scholar, 18.Grant K.M. Hassan P. Anderson J.S. Mottram J.C. J. Biol. Chem. 1998; 273: 10153-10159Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 19.Wang Y.X. Dimitrov K. Garrity L.K. Sazer S. Beverley S.M. Mol. Biochem. Parasitol. 1998; 96: 139-150Crossref PubMed Scopus (33) Google Scholar), and Crithidia (20.Brown L. Hines J.C. Ray D.S. Nucleic Acids Res. 1992; 20: 5451-5456Crossref PubMed Scopus (18) Google Scholar). The trypanosomatid CRKs have several features in common with yeast and human CDKs (16.Mottram J.C. Parasitol. Today. 1994; 10: 253-257Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 18.Grant K.M. Hassan P. Anderson J.S. Mottram J.C. J. Biol. Chem. 1998; 273: 10153-10159Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 21.Mottram J.C. Grant K.M. Biochem. J. 1996; 316: 833-839Crossref PubMed Scopus (29) Google Scholar), including 50–55% amino acid sequence identity with CDK1 from a range of organisms (15.Mottram J.C. Smith G. Gene. 1995; 162: 147-152Crossref PubMed Scopus (64) Google Scholar), a recognizable (but divergent) “PSTAIR” box, which is a domain shown to be important for cyclin binding (11.Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massagué J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1216) Google Scholar), and key threonine and tyrosine residues known to be important sites of phosphorylation in yeast and mammalian (9.Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (498) Google Scholar). The CRKs also have sequence features that distinguish them from yeast or mammalian CDKs, including an N-terminal extension for CRK2 and CRK3 (15.Mottram J.C. Smith G. Gene. 1995; 162: 147-152Crossref PubMed Scopus (64) Google Scholar) and two additional domains within the catalytic domain of the kinase for CRK4 (20.Brown L. Hines J.C. Ray D.S. Nucleic Acids Res. 1992; 20: 5451-5456Crossref PubMed Scopus (18) Google Scholar). 2J. R. Ford and J. C. Mottram, unpublished information. Hence the regulation of the trypanosome cell cycle, whose cell biology differs significantly from many other eukaryotic organisms, may also differ at the molecular level. In contrast to the CRKs, much less is known about the activating cycling partners in trypanosomes. So far only one putative cyclin gene,CYC1, has been identified (22.Affranchino J.L. González S.A. Pays E. Gene. 1993; 132: 75-82Crossref PubMed Scopus (33) Google Scholar). Despite evidence to suggest that this gene encodes a mitotic cyclin (22.Affranchino J.L. González S.A. Pays E. Gene. 1993; 132: 75-82Crossref PubMed Scopus (33) Google Scholar), other studies provide no evidence that CYC1 is a functional cyclin. 3T. C. Hammarton, J. R. Ford, and J. C. Mottram, manuscript in preparation. CYC1 may be involved, however, in regulating differentiation of the bloodstream form into the procyclic form (23.Hua S.B. Mutomba M.C. Wang C.C. Mol. Biochem. Parasitol. 1997; 84: 255-258Crossref PubMed Scopus (8) Google Scholar). Identification of cyclin genes has proven to be difficult due to their divergence at the primary amino acid level (24.Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1803) Google Scholar). Hence, it is crucial to define cyclins on a functional basis to validate their identification. In this study we used a functional complementation assay to isolate trypanosome cyclins based on their ability to rescue a S. cerevisiae G1cyclin conditional mutant. A variety of heterologous cyclins from species as divergent as Homo sapiens (25.Lew D.J. Dulic V. Reed S.I. Cell. 1991; 66: 1197-1206Abstract Full Text PDF PubMed Scopus (666) Google Scholar),Drosophila (26.Leopold P. O'Farrell P.H. Cell. 1991; 66: 1207-1216Abstract Full Text PDF PubMed Scopus (124) Google Scholar), and Arabidopsis (27.Day I.S. Reddy A.S.N. Golovkin M. Plant Mol. Biol. 1996; 30: 565-575Crossref PubMed Scopus (27) Google Scholar) have been shown to rescue cln1,2,3 lethality. This functional complementation assay enabled us to identify two novel T. brucei cyclin genes, CYC2 and CYC3, and to define the first trypanosome cyclin-dependent kinase complex. All standard DNA techniques were performed as described by Sambrook et al. (28.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast transformation was performed according to the lithium acetate procedure (29.Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Plasmid recovery from yeast used the glass bead-phenol technique (30.Hoffman C.S. Winston F. Gene. 1987; 57: 267-272Crossref PubMed Scopus (2043) Google Scholar) followed by transformation of Escherichia coli. Plasmid isolation from E. coli was performed using Qiaprep columns (Qiagen). DNA sequencing was performed using an ABI automatic sequencer. The complementation study was performed with the DL1 strain of S. cerevisiae in which the three CLN genes (CLN1, CLN2, and CLN3) were inactivated, and a chimeric gene consisting of the GAL1 promoter fused to the CLN2 protein-coding region was integrated at the LEU2chromosomal locus (25.Lew D.J. Dulic V. Reed S.I. Cell. 1991; 66: 1197-1206Abstract Full Text PDF PubMed Scopus (666) Google Scholar). The GAL1 promoter directs high level expression when the cells are grown on galactose-containing medium but is repressed if the cells are grown on a medium containing glucose. DL1 cells are able to grow on galactose-based medium but arrest in G1 on a glucose-based medium. DL1 cells were transformed with a T. brucei cDNA library, which was constructed in the yeast expression vector pRS416-met (31.Mazhari-Tabrizi R. Eckert V. Blank M. Müller R. Mumberg D. Funk M. Schwarz R.T. Biochem. J. 1996; 316: 853-858Crossref PubMed Scopus (45) Google Scholar). The pRS416 vector carries the URA3 gene as a selectable marker, and the cDNAs are fused with the MET25 promoter at the 5′ end and the CYC1 terminator at the 3′ end. Antipeptide antisera were raised against the last 9 amino acids of CRK1 (EHPYFSVEF) and the last 16 amino acids of CRK2 (EVREEEVEKLMRFNGA). Peptides were conjugated to keyhole limpet hemocyanin and used to immunize rabbits using standard procedures (32.Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Antiserum specific to CRK3 was raised by immunization of rabbits with purified recombinant CRK3 that was tagged with six histidines at its N terminus (33.Glasssmith G. Characterization of cdc2-related Kinases from Trypanosoma brucei. PhD thesis. University of Glasgow, 1997Google Scholar). The mouse monoclonal antibody BB2 (34.Bastin P. Bagherzadeh A. Matthews K.R. Gull K. Mol. Biochem. Parasitol. 1996; 77: 235-239Crossref PubMed Scopus (224) Google Scholar) was used as the anti-TY antibody. Two annealed oligonucleotides (OL49 and OL50, see TableI), which composed the TY-epitope flanked by BglII-compatible overhangs, were ligated into the uniqueBglII site near the C terminus of CYC2 (Fig. 2), after which proper integration of the TY tag was confirmed by sequence analysis. Subsequently, CYC2ty was cloned in the unique EcoRI andXhoI sites of the yeast expression vector pRS416-met (31.Mazhari-Tabrizi R. Eckert V. Blank M. Müller R. Mumberg D. Funk M. Schwarz R.T. Biochem. J. 1996; 316: 853-858Crossref PubMed Scopus (45) Google Scholar) to produce plasmid pGL387. In addition, CYC2ty was also cloned into the single Bsp120I site of the tetracycline-inducible trypanosome expression vector pHD675 (35.Biebinger S. Wirtz E. Lorenz P. Clayton C.E. Mol. Biochem. Parasitol. 1997; 85: 99-112Crossref PubMed Scopus (201) Google Scholar) to produce plasmid pGL289.Table IPrimer sequencesPrimer nameSequenceCYC2KO5′5′5′-GCATGCGGTGCAACGCGTGTGAGGA-3′CYC2KO5′3′5′-TCTAGACCGACGTTCTACTGCCACT-3′CYC2KO3′5′5′-GGTACCTTGCAGCAACGAATTGTCC-3′CYC2KO3′3′5′-GAATTCTCCCTTGTTCTTCCATTTC-3′OL115′-GGCACTGGTCAACTTGGCCA-3′OL155′-CCGTGGGCTTGTACTCGGTCA-3′OL495′-GATCTGAGGTCCATACTAACCAGGATCCACTTGACA-3′OL505′-GATCTGTCAAGTGGATCCTGGTTAGTATGGACCTCA-3′OL2435′-ACTGCTCCTGATTCGC-3′OL4345′-TCATTATCAGCGGGTAGTGTCA-3′OL4465′-GCATGCGGTGCAACGCGTGTGAGGA-3′QL2335′-GAATTCTCCCTTGTTCTTCCATTTC-3′ Open table in a new tab Procyclic form T. brucei EATRO 795 (36.Turner C.M.R. Barry J.D. Parasitology. 1989; 99: 67-75Crossref PubMed Scopus (112) Google Scholar) orT. brucei STIB 247 (37.Melville S.E. Leech V. Gerrard C.S. Tait A. Blackwell J.M. Mol. Biochem. Parasitol. 1998; 94: 155-173Crossref PubMed Scopus (94) Google Scholar) were grown at 28 °C in SDM-79 medium (38.Brun R. Schonenberger M. Acta Trop. 1979; 36: 289-292PubMed Google Scholar) containing 10% (v/v) heat-inactivated fetal bovine serum. Transfection of procyclic trypanosome cells was performed by an electroporation method. 107 procyclic trypanosomes from a mid-log phase culture were harvested and washed in transfection buffer (132 mm NaCl, 8 mm KCl, 8 mmNa2HPO4, 1.5 mmKH2PO4, 1.5 mmMg(C2H3O2)2·H2O and 90 μmCa(C2H3O2)2·H20), after which the cells were resuspended in 500 μl of transfection buffer with 5–10 μg of DNA and electroporated (1500 volts/25 microfarads). Subsequently, cells were incubated in SDM-79 medium at 28 °C for 16 h, after which antibiotics were added, and cells were cloned directly by limiting dilution. Procyclic EATRO 795 cells were transfected with the pHD449 vector for constitutive expression of the tetracycline-repressor protein (35.Biebinger S. Wirtz E. Lorenz P. Clayton C.E. Mol. Biochem. Parasitol. 1997; 85: 99-112Crossref PubMed Scopus (201) Google Scholar). Subsequently, a clonal population of these cells were transfected with the CYC2ty expression vector (pGL289, linearized with NotI), after which cells were re-cloned by limiting dilution. Induction of expression of CYC2ty was initiated in mid-log phase cultures by the addition of tetracycline (50 ng/ml), and the cells were harvested 16 h after induction by centrifugation and subsequent washing with phosphate-buffered saline (18.Grant K.M. Hassan P. Anderson J.S. Mottram J.C. J. Biol. Chem. 1998; 273: 10153-10159Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Cell pellets or protein samples were resuspended in Laemmli buffer, separated on 10 or 12.5% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes. Western blots were performed as described before (21.Mottram J.C. Grant K.M. Biochem. J. 1996; 316: 833-839Crossref PubMed Scopus (29) Google Scholar) with a 1:100 dilution of the crude antiserum directed to either CRK1, CRK2, CRK3 or with a 1:200 dilution of medium derived from cultures with the hybridoma cells producing the anti-TY antibody. Blots were performed in absence or presence of blocking peptide (2 μg/ml). Antigens were detected using a chemiluminescence detection system (Pierce SuperSignal). Leishmanial p12cks1 or yeast p13suc1 protein was coupled to Amino-link beads at a concentration of 5 mg ml−1 as described previously (21.Mottram J.C. Grant K.M. Biochem. J. 1996; 316: 833-839Crossref PubMed Scopus (29) Google Scholar). Selection of T. brucei CRKs was carried out as described for the Leishmania mexicana CRK (18.Grant K.M. Hassan P. Anderson J.S. Mottram J.C. J. Biol. Chem. 1998; 273: 10153-10159Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Briefly, 108cells were lysed in 400 μl of lysis buffer (50 mm MOPS, pH 7.2, 100 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm sodium orthovanadate, 10 mm NaF, 10% glycerol, and 1% Triton X-100) containing a mixture of protease inhibitors (2.5 mm 1,10-phenanthroline, 100 μg ml−1 leupeptin, 5 μg ml−1pepstatin A, and 500 μg ml−1 Pefabloc® SC). Lysates were incubated on ice for 15 min and then centrifuged at 100,000 × g for 45 min at 4 °C. The resulting supernatants (S-100 lysates) were incubated for 2 h with 40 μl of either control or p12cks1 or p13suc1 beads (18.Grant K.M. Hassan P. Anderson J.S. Mottram J.C. J. Biol. Chem. 1998; 273: 10153-10159Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), after which the beads were extensively washed with lysis buffer and used for immunoblotting or histone H1 kinase assays. Immune-precipitations and kinase assays were performed as described previously (17.Mottram J.C. Kinnaird J. Shiels B.R. Tait A. Barry J.D. J. Biol. Chem. 1993; 268: 21044-21051Abstract Full Text PDF PubMed Google Scholar, 21.Mottram J.C. Grant K.M. Biochem. J. 1996; 316: 833-839Crossref PubMed Scopus (29) Google Scholar). S-100 cell extracts, prepared as described above, were incubated with 50 μl of serum in the absence or presence of 5 μg ml−1 peptide for 60 min at 4 °C. Subsequently, 25 μl of protein A-Sepharose (Amersham Pharmacia Biotech) was added and incubated for a further 30 min at 4 °C. After extensive washing of the Sepharose beads with lysis buffer, the beads were either resuspended in Laemmli buffer and used for SDS-polyacrylamide gel electrophoresis and immunoblotting or resuspended in kinase assay buffer (50 mm MOPS, pH 7.2, 20 mm MgCl2, 2 mm dithiotheitol, 10 mm EGTA, 250 μg ml−1 histone H1 (Life Technologies, Inc.) and 4 μm ATP (0.1 μCi of [γ-32P]ATP) and incubated for 30 min at 30 °C for a histone H1 kinase activity assay (21.Mottram J.C. Grant K.M. Biochem. J. 1996; 316: 833-839Crossref PubMed Scopus (29) Google Scholar). Kinase activity of SDS-polyacrylamide gel electrophoresis-separated samples was examined by drying of the gel and exposure to an x-ray film. A yeast two-hybrid interaction assay was performed using the LexA Hybrid Hunter system (Invitrogen).T. brucei CRK1 and CRK3 were cloned into theEcoRI/PstI site of pHybLex/Zeo by constructing unique EcoRI and PstI sites on the 5′ and 3′ ends of the coding region by PCR. The inserts for CRK1 (pGL176) and CRK3 (pGL177) were sequenced to confirm in-frame fusions with LexA and to check that no PCR mutations had been introduced. TheCYC2 and CYC3 genes were cloned into theHindIII/StuI sites of the pYESTrp vector using the same PCR-based approach to give plasmids pGL182 and pGL181, respectively. These produced fusions with the B42 domain. Bait and prey plasmids were transformed sequentially into S. cerevisiaestrain L40 (Invitrogen), and protein:protein interactions were assessed by β-galactosidase activity using a filter lift assay as described by the manufacturer. Expression of the fusion proteins was checked by Western blot analysis using antibodies (Invitrogen) against LexA and the V5-epitope, which is fused to the B42 domain, according to the manufacturer's protocol. In the absence of a sufficient 5′ and 3′ sequence flanking the CYC2 gene, a gene disruption approach was adopted to knock out CYC2 gene function rather than a conventional gene deletion. The CYC2 gene was disrupted by transfection with BLE and PACresistance knockout constructs. These constructs were designed to replace 160 bp of sequence in the middle of the CYC2 open reading frame with the required antibiotic resistance gene. Correct transcription and post-transcriptional processing of the drug resistance genes relied on 5′ and 3′ PARP control sequences. A 320-bp DNA fragment containing the 5′ end of CYC2 was amplified from plasmid pGL163 with primers CYC2KO5′5′ (See Table I for primer sequences) and CYC2KO5′3′ and cloned into pCR-script. A 320-bp DNA fragment containing the 3′ end of CYC2 was amplified from plasmid pGL163 with primers CYC2KO3′5′ and CYC2KO3′3′ and also cloned into pCR-script. CYC2KO5′5′ and CYC2KO5′3′ were engineered withSphI and XbaI sites, respectively, whereas CYC2KO3′5′ and CYC2KO3′3′ were engineered with KpnI andEcoRI sites, respectively. pBluescript-based plasmid pGL110 containing the 5′-PARP-PAC-3′-PARP cassette (derived from pJP44 (39.Sherman D.R. Janz L. Hug M. Clayton C. EMBO J. 1991; 10: 3379-3386Crossref PubMed Scopus (98) Google Scholar)) was used to make the PAC-resistant CYC2 deletion constructs. The 320-bp KpnI/EcoRI fragment containing theCYC2 3′ end was cloned intoKpnI/EcoRI-digested pGL110. Subsequently, theCYC2 5′ flank was cloned intoSphI/XbaI sites to give pGL2. The BLE-resistant cassette (pGL1) was generated by removing the 1.7-kb 5′-PARP-PAC-3′-PARP fragment by digestion withXbaI/KpnI and replacing with the 1.5-kbXbaI/KpnI 5′-PARP-BLE-3′-PARP fragment from pGL108. The disruption cassettes were excised by digestion withSphI and EcoRI, and the insert was gel-purified using a Qiextract kit (Qiagen). Approximately 5 to 10 μg of DNA was used for each transfection with 4 × 107 mid-log procyclic STIB 247 trypanosomes, after which transfected parasites were cloned by limiting dilution. PCR analysis on integration of the CYC2 disruption cassettes was performed on chromosomal DNA prepared by a mini-prep procedure (40.Medina-Acosta E. Cross G.A.M. Mol. Biochem. Parasitol. 1993; 59: 327-329Crossref PubMed Scopus (234) Google Scholar). Distinct PCR experiments were performed in which five oligonucleotides in total were used (Table I, see also Fig.8 A). Logarithmic phase procyclic form trypanosomes were induced to express CYC2ty by the addition of 1 μg ml−1 tetracycline. Cells were harvested, air-dried smears prepared, and slides were fixed by immersion in −20 °C methanol for at least 30 min. After fixation, slides were rehydrated in phosphate-buffered saline for 5 min and then incubated with the BB2 monoclonal antibody for 45 min. After washing, anti-mouse fluorescein isothiocyanate conjugate diluted 1:50 was applied to the slides for a further 45 min, and the cells were washed and counterstained with 4,6-diamidino-2-phenylindole (1 mg/ml) and mounted in MOWIOL (Harlow Chemical Co., UK) containing 1 mg ml−1 phenylenediamine as an anti-fading agent. Cells were visualized on a Zeiss Axioscope microscope, and images were captured and processed using a Hamamatsu CCD camera, NIH image 1.58, and Adobe Photoshop 5. The low level of sequence identity between cyclins of different species precludes in general the use of PCR-based approaches to identify cyclins. Hence, we used a functional complementation assay to isolate trypanosome cyclins based on their ability to rescue aS. cerevisiae G1/S phase cyclin conditional mutant. Yeast DL1 cells were transformed with a T. bruceicDNA library cloned in the yeast expression vector pRS416-met (31.Mazhari-Tabrizi R. Eckert V. Blank M. Müller R. Mumberg D. Funk M. Schwarz R.T. Biochem. J. 1996; 316: 853-858Crossref PubMed Scopus (45) Google Scholar). 5 × 105 cells were plated on solid medium lacking uracil but containing glucose, and 200 transformed colonies were obtained. Of these 157 were plasmid-independent revertants that had lost the plasmid and were able to grow on glucose plates containing uracil and the antimetabolite 5-fluoro-orotic acid. The 43 remaining plasmids were recovered in E. coli and used to transform the DL1 yeast strain a second time. Transformants obtained with six of the plasmids were able to grow on the glucose-based medium, showing that these plasmids were able to complement the cyclin deficiency (Fig.1). The six cDNAs were grouped into two classes by restriction analysis and partial sequencing. The two different cDNAs of 870 bp (pGL163) and 1.79 kb (pGL164) were then sequenced, and the genes were named CYC2 andCYC3. CYC2 is predicted to encode a 24.3-kDa protein (CYC2) of 211 amino acids, with an isoelectric point of 6.82. CYC3 is predicted to encode a 46.5-kDa protein (CYC3) of 414 amino acids, with an isoelectric point of 7.24. Computer searches of data bases with the CYC2 and CYC3 protein sequences found significant homology to a wide range of cyclins. This homology is, however, restricted to a stretch of about a 100 amino acids known as the cyclin box, which is involved in binding of the CDK partner (9.Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (498) Google Scholar). The cyclin box of CYC2 has 38% identity with that ofNeurospora crassa PREG1 regulatory protein (41.Kang S. Metzenberg R.L. Genetics. 1993; 133: 193-202Crossref PubMed Google Scholar), 25% identity with the cyclin box of S. cerevisiae PHO80 cyclin (42.Kaffman A. Herskowitz I. Tjian R. O'Shea E.K. Science. 1994; 263: 1153-1156Crossref PubMed Scopus (316) Google Scholar), and 22% identity with the cyclin box of human cyclin D3 (43.Motokura T. Keyomarsi K. Kronenberg H.M. Arnold A. J. Biol. Chem. 1992; 267: 20412-20415Abstract Full Text PDF PubMed Google Scholar) (Fig. 2 B). The cyclin box ofCYC3 has 32% identity with S. cerevisiae CLB1 (44.Surana U. Robitsch H. Price C. Schuster T. Fitch I. Futcher A.B. Nasmyth K. Cell. 1991; 65: 145-161Abstract Full Text PDF PubMed Scopus (350) Google Scholar) and 32% identity with CLB2 (45.Richardson H.E. Lew D.J. Henze M. Sugimoto K. Reed S.I. Genes Dev. 1992; 6: 2021-2034Crossref PubMed Scopus (208) Google Scholar) (Fig. 2 C). Apart from the cyclin boxes, the other regions of CYC2 and CYC3 demonstrate no significant homology to any other known protein, although CYC3 does contain a destruction box-like motive (RGTLVVPRN, see F
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