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Isolation of a mitotic-like cyclin homologue from the protozoan Trypanosoma brucei
Gene, 132 (1993) 75-82 0 1993 Elsevier Science Publishers
GENE
B.V. All rights reserved.
0378-l 119/93/$06.00
0729 1
Isolation of a mitotic-like cyclin homologue from the protozoan Trypanosoma brucei (Cell cycle; cDNA cloning; polymerase chain reaction; c&2; M-phase kinase; mitosis; destruction box)
JosC L. Affranchino, Silvia A. Gonzalez and Etienne Pays Department of Molecular
Biology, University of Brussels, 1640, Rhode St. Get&e. Belgium
Received by J.R. Kinghorn:
24 November
1992; Revised/Accepted:
5 April/l5
April 1993; Received at publishers:
27 May 1993
SUMMARY
Activation of the p34cdc2protein kinase (PK) at different stages of the eukaryotic cell cycle is controlled by interaction with regulatory proteins known as cyclins (CYCs). Using a probe obtained by PCR amplification, we have isolated from the protozoan, Trypanosoma brucei, a cDNA clone encoding a CYC homologue. The amino acid sequence deduced for this gene (CYCI) shares structural homology with A- and B-type CYCs of other organisms, including a motif, the destruction box, which has been related to the rapid turnover of these CYC proteins in mitosis. When expressed in fission yeast, CYCZ is able to rescue the defect of a temperature-sensitive cdcl3 mutant, demonstrating that it is functional as a cell-cycle regulator. In trypanosome cells, CYCl associates with a 34-kDa protein that cross-reacts with a monoclonal antibody against the conserved ‘PSTAIR’ epitope of p34cdc2, and the complex displays histone Hl PK activity. Furthermore, when trypanosome cells are synchronized by hydroxyurea treatment, CYCl accumulates as cells progress towards mitosis. These observations, taken together, suggest that CYCl is a component of the active PK complex required for the control of trypanosome mitosis.
INTRODUCTION
The eukaryotic cell cycle is controlled at two major decision points, the transitions GljS and G2/M, by the activity of a 34-kDa serine-threonine PK which is the product of the cdc2 gene (Nurse and Bisset, 1981). It is Correspondence to: Dr. J.L. Affranchino at his present address: Centro de Virologia Animal (CEVAN), Serrano 661 (1414), Buenos Aires, Argentina. Tel. (54-l) 857 0012; Fax (54-l) 856 4495. Abbreviations: aa, amino acid(s); bp, base pair(s), BSA, bovine serum albumin; cDNA, DNA complementary to RNA; CYC, cyclin; CYCI, gene encoding CYCl; DMSO, dimethylsulfoxide; kb, kilobase or 1000 bp; mAb, monoclonal antibody(ies); nt, nucleotide(s); ORF, open reading frame: oligo, oligodeoxyribonucleotide; PBS, phosphatebuffered saline (0.137 M NaC1/0.0027 M KCl/0.0043 M Na,HP0,/0.0014 M KH,PO,); PCR, polymerase chain reaction; PK, protein kinase; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/O.OlS M Nasecitrate pH 7.6; Sz.. Schizosaccharomyces; T., Trypanosoma; ts, temperature sensitive.
thought that the p34’*” PK phosphorylates a variety of substrates that are directly involved in the commitment of the cell to the events of chromosomal replication and mitosis (Moreno and Nurse, 1990). The activity of p34 PK depends on the dephosphorylation of Tyr and Thr residues by a phosphatase encoded by the cdc25 gene (Gautier et al., 1991; Dunphy and Kumagai, 1991). In addition to these post-translational modifications, regulatory proteins known as CYCs, that associate with ~34, confer kinase activity to the resulting complex (Draetta et al., 1989; Gautier et al., 1990). CYCs can be classified into two major groups on the basis of their accumulation and functional role during specific phases of the cell cycle and the presence of conserved motifs within their aa sequence. One group includes mitotic CYCs, consisting of A and B classes, that exhibit maximal levels at the G2/M transition and rapidly decrease during M phase. This abrupt decline allows cells to complete mitosis. They have been described in a vari-
76
ety of organisms including yeast (Hagan et al., 1988; Booher and Beach, 1988; Ghiara et al., 1991) sea urchin (Pines and Hunt, 1987), frog (Minshull et al., 1990), fly {Lehner and O’Farrell, 1990), and man (Fines and Hunter, 1989). The second group corresponds to CYCs that are required by the cells for passage through GljS transition to undergo DNA replication. They accumulate during Gl and are degraded in S phase (Xiong and Beach, 199 1). Although extensive information has been gathered on yeast, invertebrates and vertebrates, nothing is known about cell cycle regulation in Protozoa. Trypanosomes are flagellated parasites which alternate between an insect vector and a mammalian host. Their life cycle shows transformations from dividing into non-dividing forms, offering an attractive model for cell division control studies. Proliferating forms are essential for the development of an infection, and differentiation to non-dividing forms is necessary for transmission between hosts. Therefore, knowledge of the genes and proteins involved in these processes might provide novel approaches to the control of infection and parasite proliferation in the human host. The aim of this paper was to isolate from Trypanosoma brucei a gene (CYCf) encoding a polypeptide structurally and functionally related to mitotic CYCs of other organisms.
RESULTS AND DISCUSSION
(a) Isolation of a cDNA clone encoding a T. brucei cyclin (CYC) homologue
A single degenerate oligo was designed utilizing the most conserved regions of published aa sequences of CYCs (Minshull et al., 1990) and considering the positional nt preferences of trypanosomes to limit the redundancy, and used as antisense primer in PCR amplifications. To devise the sense primer, we took advantage of the fact that trypanosomes bear the same 39-nt sequence (miniexon) at the 5’ end of their mRNAs (Boothroyd and Cross, 1982). When these primers were used in PCR experiments, a DNA fragment of 500 bp was consistently obtained from cDNA of procyclic forms of T. brucei. The amplified fragment was used in turn as radiolabelled probe to screen a h gt IO-cDNA library prepared from procyclic poly(A)‘RNA. This allowed the isolation of a cDNA clone (referred to as CYCI) which was sequenced. CYC’Zwas found to be a full-length gene since it extended from the miniexon sequence present at its 5’ end to a poly(A) tail at its 3’ end (Fig. 1). The isolated gene comprises an ORF of 334 aa which predicts a polypeptide of 36 27 1 Da (Fig. 1). The primary structure of the predicted ORF of CYCl
was found to have homology to previously characterized mitotic CYCs. CYCI contains a region of similarity to the cyclin box of A- and B-type CYCs, including sequence features expected to be conserved (Fig. 2). The alignment of CYCl with these CYCs in the cyclin-box region revealed 28% identity to sea urchin CYC B, 25% to human CYC Bl and fission yeast Cdcl3, and 26 % to both human and clam CYCs A (Fig. 2). For comparison, human CYCs A and B show 46% identity over the same region. If aa substitutions considered to be conservative are allowed, a higher degree of homology (46649%) is observed for all the comparisons, The degree of relatedness between mitotic CYCs and CYCl in the region shown in Fig. 2 is statistically significant as inferred from a statistical analysis based on the method of Dayhoff et al. (1983). Alignment scores of 5.6-5.8 standard deviations above the mean were obtained with respect to comparisons involving 100 randomly shuffled versions of the CYC 1 sequence. Although CYCl cannot be readily assigned to either A- or B-type, we assume that CYCl is most likely a mitotic CYC on the basis of the presence of a destruction box and its timing of expression during trypanosome cell cycle (see section e below). In CYCs A and B, the cyclin box is located after a long N-terminal domain. The aa sequence of the N terminus is highly variable, but a conserved motif involved in the programmed mitotic proteolysis of these proteins is embedded in this area (Glotzer et al., 1991). This region, called the destruction box, consists of the partially conserved sequence RXXLXXIXN followed by a Lys-rich region which provides potential sites for ubiquitination. Remarkably, CYCl, while lacking a divergent long N-segment (the homology with the cyclin box of mitotic CYCs starts at aa 60) shows in its C-terminal region a motif which clearly conforms to the destruction box (Fig. 1). The region corresponding to residues 255 263 (RXXVXXIXN) bears a single conservative aa substitution with respect to the consensus sequence and is followed by several scattered Lys residues (Fig. 1). Therefore, a destruction box which is a hallmark of mitotic CYCs, is also present in CYCl sequence, albeit located at the C-terminal segment. (b) Genomic organization
and transcription of CYCZ To examine the organization of CYCl in T. brucei
genome, a Southern blot analysis was performed. A radioIabelled probe spanning the complete ORF of CYCl was prepared and hybridized to restriction enzyme digestions of genomic DNA of procyclic forms of T. brucei. The results showed that CYCl is encoded by a single copy gene (Fig. 3A). The detection of two or three reacting bands in HindIII, PstI and Sal1 digests correlates with the presence of either one (Hind111 and &I) or two sites
77 GCGCACTGCATCTTGTAGCACGATGATGACAAACTTGAATTCCCC WMTNLNVRTIKGCFP
GGTTCCTTACCTGATGCACTCMCGACATGTCCGTCATCAG GSLPDALNDMSVINHILSLTGRFDEGQAAL
CTTCAGCACTTAGCGGAGGCACATATCATTTTACTACTACCGGAGGTMCACCCTTTTCG~ATACGACG~GGGAGG-CGGGCCTCGA~
LQHLAEAHllLLPEVTPFSLYDAGRKRASM CGTGTCTCCGGGCI\AGTTGCAGCTCGTGGTGTTGTCTTACCCC GQVAARGVVLVGGGCCFRAEHSDSANP R" s -CATGCGCACAGTACATGTTGTCGCGCGGG-~~GTGGATTCAGGGCGGCCGG~G-TGG*G~G~GGTGCG~GT~GAG K T CAQYHLSRENEVDSGRPVEMEEGGAKWE TACCTATGCGTTCACGGCCTCAGTGTGTTGCCGGGTATAT Y LCVHGLSVLPG~FCPQHSSRDATGLLLNE AGCTTTAGTAAGATGCTGACGGCATCCAACTGAGCGAGCGAGGGATTGGTGTCGACTGCAGGGCGGTG-CTG~GATGGGTGACGGTCGG SFSKMLKRHPTERGIGVDCRAVLLL~C TACCAGGTGCTGACGATAGCCAATAGAGAGAGAACTGT AN R E G Y 0" L T I
0
G
_R
RTASV~DINIQI~DVVEGN
GTGCAGACAACCACAATTCAC~CAGGGAAGCGTAGMG~~GC~CG-CCGTGTGGACCTGTCGTACGAGACCC~TTGAGGCG
v
PVVRDPFEA
OTTTIQQQGSVEELLRXPCG
TACTATGCRATGGCCMCCC~CTG~~MCAG~G~A~ATGTGCACCCA~TGATA~GG~GAGCCAG~~CGTTAGGCC Y Y A MAN PTALTEELLCAPR* CCGGTGTACGGACATTTTGGTAGCAGTGTCGTTTGAACGnGT TTGGAATCGACACATGTGCGCGCGTTCGTCAACATGTGAACTACAG (A)63
Fig. 1. Nucleotide sequence of CYCI cDNA and deduced aa sequence. The mini-exon sequence is boxed and the stop codon of the ORF is indicated with an asterisk. The cyclin destruction box and the Lys residues which follow this motif are underlined. Methods: The oligos used in PCR amplifications (Saiki et al., 1985) were as follows: sense primer, S-GTTTCTGTACTATATTG, corresponds to the short sequence of the mini-exon which is added to the 5’ end of all trypanosome mRNAs (Boothroyd and Cross, 1985); antisense primer, S-RGYVACDCCVACNA (R = A or G; Y = C or T; V =A or C or G; D= A or G or T; N= A or C or G or T) corresponding to aa LVGVT or LVGVA, was synthesized according to a highly conserved region of the cyclin box. cDNA was prepared from poly(A)+RNA of procyclic forms of T. brucei according to the method of Gubler and Hoffman (1983). cDNA (200 ng) was used as template in 100 pl reactions containing 10 mM Tri%HCl pH 8.3/50 mM KCl/l.5 mM MgClJ0.2 mM of each dNTPj5 pM of each primer/2.5 units of Taq polymerase (BRL). After an initial denaturing step of 5 min at 95°C amplification was allowed to proceed for 35 cycles (1 min at 95”C, 2 min at 38°C and 3 min at 72°C). The PCR product was gel purified and cloned into the SmaI site of pUCl9 plasmid vector. A radiolabelled probe made from the PCR clone was used to screen a hgtlO-cDNA library prepared from poly(A)+RNA of T. brucei procyclic forms. DNA from positive phage plaques was purified and the inserts subcloned into the EcoRI site of pUCl9. The nt sequence determination was performed on both strands by the dideoxynucleotide method (Sanger et al., 1977) using a modified T7 DNA polymerase (Pharmacia). Unidirectional deletions of the cloned DNA fragments were made with exonuclease III (Henikoff, 1984). At least three independent full-length cDNA clones were completely sequenced. DNA and protein sequences were analysed using computer programmes supplied by Genetic Computer Group, Inc. (GCG package version 7.1; Devereux et al., 1984). The nt sequence has been submitted to the EMBL database with the accession No. X65625.
(MI) for these restriction enzymes within CYCZ gene sequence. To assess the steady-state level of CYCl gene transcript in the proliferating stages of T. brucei life cycle, a Northern blot of poly(A)+RNA of procyclic and bloodstream forms was hybridized to the CYCZ probe. A single transcript of 1.4 kb was detected in both forms of the parasite (Fig. 3B). The size of the hybridizing RNA band is in good agreement with the length of the corresponding cDNA clone. (c) Trypanosome CYCI gene rescues fission yeast c&13’” mutation The structural similarity between CYCl and its counterpart in fission yeast suggests that these proteins might be functionally homologous. To examine whether the CYCI gene could provide the functions carried out by cdcl3, we determined if the trypanosome gene could
rescue a fission yeast cdcl3’” mutation. CYCI cDNA was subcloned into the pAALN vector and transformed into Schizosaccharomyces pombe ade6-210 cdcl3-117 leul-32 ts mutant. Leu+ transformants were selected either at the permissive (25°C) or the restrictive temperature (35°C). Fig. 4 shows that the CYCI gene could rescue the cdcl3 mutation. At least ten independent Leu+ transformants containing the CYCl gene were able to grow at the restrictive temperature (data not shown). These data establish that CYCl is able to function as a mitotic inducer in fission yeast and suggests that CYCl is likely to control mitosis in trypanosome cells. (d) CYCl associates to a CdcZ-related polypeptide To characterize the trypanosome CYCZ gene product, antiserum against the bacterially expressed protein was prepared, affinity purified and used to probe a Western blot of protein extracts of procyclic forms. The CYCI
kb
A
B
abcde
pb
I)
Fig. 2. Comparison of trypanosome CYCl with A- and B-type CYCs in the cyclin box region. (A) The protein encoded by CYCI (Tb) was aligned with human CYC A (HU A; Wang et al., 1990) and clam CYC A (Cl A, Swenson et al., 1986). The alignments of the three sequences are based on the outputs of pairwise alignments assisted by the BESTFIT program (Devereux et al., 1984). Boxes indicate identical residues or conserved aa substitutions (as assigned by the program BESTFIT) between CYCl and the other CYCs. Closed and open circles designate, respectively, the positions of identical and similar aa in CYCl sequence with respect to those most likely to be conserved in A-type CYCs. (B) Alignment of CYCl (Tb) with sea urchin CYC B (SU; Pines
- 1.4 kb
Fig. 3. Genomic organization and transcription of the trypanosome CYCI gene. (A) Genomic DNA of procyclic forms of T. brucei was digested
with BnmHI
(lane a), EcoRI (lane b), Hind111 (lane c), PstI
(lane d) and Sal1 (lane e), resolved on a 0.85% agarose gel and blotted onto a nitrocellulose membrane. The filter was hybridized to a CYCI radiolabelled probe. (B) Identification of a CYCl gene transcript. A Northern blot of poly(A)+RNA purified from bloodstream (b) and procyclic forms (p) of T. brucei was hybridized to a CYCI probe. Methods: Trypanosoma brucei bloodstream forms (AnTat 1.3A clone) and procyclic forms (AnTat l.lB clone) were used in these experiments. High molecular weight genomic DNA purification and Southern blotting
and Hunt, 1987), human CYC Bl (HU; Pines and Hunter, 1989) and fission yeast Cdcl3 (Booher and Beach, 1988; Hagan et al., 1988). Closed
were performed as previously described (Affmnchino et al., 1991). Total RNA was purified by the urea-LiCl method (Auffray and Rougeon, 1980). Poly(A)‘RNA (2 ug) was denatured by glyoxal-DMSO treatment
and open circles indicate, respectively, the positions of CYCl with identical and similar aa to those highly conserved in B-type CYCs.
(Thomas, 1980), resolved by electrophoresis and blotted onto nitrocellulose membranes
antiserum identified a polypeptide of an apparent molecular size of 36 kDa, which agrees with the predicted size calculated for the primary translation product of CYCI (Fig. 5A). Similar protein blots probed with the corresponding preimmune serum did not display any detectable signal (data not shown). To analyze whether a ~34’~” homologue exists in trypanosomes, a protein blot was probed with a mAb against the PSTAIR epitope present in all Cdc2 proteins described so far. An immunoreactive band of 34 kDa was clearly detected (Fig. 5A). This result prompted us to investigate whether CYCl associates with the identified Cdc2 homologue. An asynchronous culture of procyclic forms was metabolically labelled with [35S]methionine and parasite cell lysates were immunoprecipitated with CYC 1 antiserum. The anti-CYC 1 immunocomplexes showed, in addition to the CYCl band, a polypeptide with the expected electrophoretic mobility for the putative Cdc2 homologue (Fig. 5B).
on 1.5% (w/v) agarose gel (Thomas, 1980). All hybrid-
izations were carried out in 3 x SSCj5 x Denhardt’s solution/O. 1% (w/v) SDS/SO ug/ml salmon sperm DNA for 16 h at 65°C. Filters were washed three times at 65°C in 0.1 x SSC/O.l% SDS.
None of these bands were observed when immunoprecipitations were performed with preimmune serum (Fig. 5B). These results show that CYCl antiserum coimmunoprecipitates a protein of 34 kDa, presumably p34’d’2. To verify that this 34-kDa polypeptide was indeed the Cdc2 homologue, anti-CYCl immunoprecipitates were Western blotted and reacted with the PSTAIRspecific mAb. Fig. 5C shows that an immunoreactive band of 34 kDa was observed in CYCl immunocomplexes but could not be detected in immunoprecipitates prepared with preimmune serum. Taken together, these observations demonstrate the existence of a physical association between CYCl and a polypeptide antigenically related to ~34’~‘~. To determine whether these complexes exhibit PK activity, anti-CYCl immunoprecipitates were examined
79
Fig. 4. Rescue of the Sz. pombe cdcl3 ts mutation. Sz. pombe strain ade6-210 cdcJ3-117 Ieul-32 was transformed with the expression vector pAALN (c), the plasmid pAALN carrying the CYCZ cDNA in the correct transcription orientation (b) and pAALN containing CYCl in the reverse transcription orientation (d). Leu+ transformants (b-d) and the parental strain (a) were streaked on minimal medium plates and incubated either at the permissive (25°C) or restrictive temperature (35°C). Cells were allowed to recover for 24 h at 25°C before shifting to the restrictive temperature. The plates were photographed after 5 days of incubation. Methods: The complete trypanosome CYCI cDNA was cloned in the correct transcription orientation into the EarnHI site of the pAALN vector (Xu et al., 1992) containing the LEU2 gene as selectable marker. The plasmid was transformed into Sz. pombe ade6-210 cdcl3-117 leul-32 ts strain (SP731) by the Li.ace.tate method (Moreno et al., 1991). Leu+ transformants were isolated at 25°C by plating on Edinburgh minimal medium plates (Moreno et al., 1991) supplemented with adenine. As controls, cells were transformed with the pAALN vector alone and a plasmid containing the CYCl gene in the reverse transcription orientation.
for their ability to phosphorylate histone H 1. As expected, the immunocomplexes obtained with serum against CYCl displayed a specific histone Hl PK activity, while the control immunocomplexes (preimmune serum) failed to catalyze the phosphorylation of this substrate (Fig. 5D). Therefore, CYCl seems to exert its biological function by integration with ~34’~” into an enzymatically active PK complex. (e) CYCl levels are cell-cycle regulated A distinctive characteristic of CYCs is their oscillation through the cell cycle showing accumulation and destruction at defined points (Pines and Hunter, 1989). We therefore sought to determine whether the level of CYCl varies during the cell cycle. To this end, an exponentially growing culture of procyclic forms of T. brucei was synchronized by incubation with hydroxyurea for 9 h and released from the arrest by inoculation into fresh medium. Hydroxyurea treatment is the only method proved to be efficient for the synchronization of trypanosome cells 1969; Simpson (Steinert, and Braly, 1970). Synchronization was monitored by analysis of DNA synthesis and quantification of cells in late G2/M (Fig. 6A). Parallel samples were taken at different time intervals to prepare protein and RNA samples. Reaction of protein blots with CYCl antiserum and the PSTAIR-specific mAb allowed us to assess the protein levels of CYCl and p34’d” in each sample (Fig. 6B). The results showed that CYCl level began to rise after S phase completion peaking 6-7.5 h after release from the arrest (Fig. 6B). By contrast, ~34’~‘~was detected at constant levels throughout the experiment (Fig. 6B). To examine whether accu-
mulation of the CYCl gene transcript contributes to the regulation of CYCl polypeptide abundance, a Northern blot analysis was performed. Fig. 6C shows that the CYCl mRNA level increased as cells progressed towards mitosis, which correlates with the timing of accumulation of the corresponding protein. The pattern observed for the expression of CYCl suggests that it is required during GZ/M phase and that the availability of this CYC could be controlled at least in part by the regulated accumulation of the CYCZ gene transcript. (f) Conclusions We have isolated from T. brucei a gene encoding a polypeptide homologous to CYCs. The results presented herein constitute the first evidence for the existence of these cell cycle regulators in Protozoa. Four sets of evidence suggest that CYCl represents the trypanosome counterpart of the CYCs of other organisms: (I) The primary structure of CYCl shows significant similarity, indicative of structural homology, to mitotic CYCs. CYCl is less closely related to A- and B-type CYCs than they are to each other, which suggests that CYCl may represent a novel mitotic type or a highly diverged version of A- and B- CYCs. Regarding the structural divergence of CYCl, distinctive features of trypanosome protein sequences, such as peptide insertions within the region of homology with proteins of other organisms, have been well documented (Alexandre et al., 1990; Revelard and Pays, 1991). Besides, the identification of distantly related Gl CYCs in different organisms, such as human CYC C (Lew et al., 1991) and yeast CLN proteins (Hadwiger et al., 1989), supports the notion that in
80
c P36
cyc1 -o-p34 -
P34
1
2
1
2
1
2
1
histone Hl
2
Fig. 5. CYCl polypeptide forms a complex with a p34Cdczhomologue that phosphorylates histone HI in vitro. (A) Characterization of anti-CYCl antibodies. Western blots of protein extracts prepared from procyclic forms of T. brucei were incubated with affinity-purified Ab against CYCl (lane I), and a PSTAIR-specific mAb (lane 2). The molecular mass markers (kDa) are indicated on the left margin. (B) CYCI antiserum co-immunoprecipitates a 34-kDa polypeptide. Lysates prepared from [3sS]methionine-1abell~ parasites were immunopre~ipitat~ with either preimmune serum (lane 1) or anti-CYCl serum (lane 2). Immunocomplexes were resolved on a 0.1% SDS-lo% polyacrylamide gel (Laemmli, 1970) and autoradiographed. (C) The 34-kDa polypeptide co-immunoprecipitated by anti-CYCl antibodies is antigenically related to ~34’~“. Parasites were lysed and immunoprecipitated with either preimmune serum (lane 1) or anti-CYCl serum (lane 2). Immunocomplexes were resolved on a 0.1% SDS-12% polyacrylamide gel, transferred onto nitro~1lulo~ filters and reacted with the PSTAIR-s~cific mAb. The portion of the blot containing IgG was removed. (D) AntiCYCl immunoprecipitates exhibit histone Hl PK activity. Immunocomplexes prepared either with preimmune serum (lane 1) or anti-CYCl serum (lane 2) were examined for their ability to catalyse the phosphorylation of histone Hl. Methods: To obtain an immunogen for anti-CYCl antibodies production, the complete ORF of CYCI cDNA (bp 46-1078) was amplified by PCR and cloned into the EcoRI site of pKK223-3 plasmid vector (Pharmacia). This construct allowed the expression of the CYCt gene from its own initiating ATG. The recombinant polypeptide was expressed in Escherichiu cofi (JM109 strain) and isolated by electroelution from a preparative 0.1% SDS-12% polyacrylamide gel. Specific Ab against CYCI were raised in white New Zealand rabbits. The antiserum against CYCl was affinity purified by adsorption to and elution from nitrocellulose strips containing bacterially expressed protein (Smith and Fisher, 1984). Antibodies were eluted from the nitrocellulose with 0.1 M glycine pH 2.4 and immediately neutralized with one tenth volume of 1 M TrisHCl pH 8.0. Immunoblotting was performed essentially as described in Affranchino et al. (1989). Protein lysates from procyctic cells were resolved on 0.1% SDS-12% ~lyacrylamide gels and blotted onto nitrocellulose membranes. Filters were incubated either with affinity-purified anti-CYCl Ab (diluted 1:lOO)or with the PSTAIR-specific mAb (200 ng/ml). Antigen-antibody reaction was developed by incubation with i251-labelled Protein A (> lo7 dpm/pg, 5 x lo5 cpm/ml; Amersham). Rabbit anti-mouse serum (400 ng/ml) was included in the incubation mixture in the case of the membrane reacted with the mAb. For immunoprecipitations procyclic forms grown to a cell density of lO’/ml were starved for 2 h in methionine-deficient Cunningham medium (Cunningh~, 1977) and metabolically labelled for 2 h with 100 uCi/ml [3sS]methionine (1120 Ci/mmol, ICN Biomedicals). Parasites were harvested, washed twice in cold PBS and lysed at 0°C with 300 ~1 Lysis buffer (50 mM TrisHCl pH 8.0/150 mM NaCl/l% [w/v] NP-40/10 mM NaF/lOO uM NaV0,/60 mM B-glycerophosphate/S mM EDTA/300 pM PMSFjlO ug/ml trasylol/5 pg/ml leupeptin/l kg/ml pepstatin). After 30 min at O”C, lysates were spun at 12000 x g for 10 min and the resulting supernatants subjected to imm~oprecipitation assays. Lysates (100 ul) were precleared by the addition of 30 pl of protein A-Sepharose CL4B beads (Pharmacia) 10% (w/v) in lysis buffer + 1% (w/v) BSA. Samples were rotated end-over-end for 30 min at 4°C and centrifuged 1 min at 12000 x g. The supernatants were incubated with 1 ul of anti-CYCl serum for 2-4 h at 4°C. As control, lysates were immunoprecipitated with preimmune rabbit serum. Subsequently, a 30 ul sample of protein A-Sepharose beads 10% (w/v) in lysis buffer was added and the mixture rotated end-over-end during 1 h at 4°C. Beads were then pelleted and washed three times in lysis buffer + 0.1% (w/v) BSA. The immunocomplexes were released by boiling in 60 ~1 of 1 x Laemmli’s sample buffer (Laemmli, 19’70),resolved on a 0.1% SDS-IO% polyacrylamide gel and autoradiographed after fluorographic treatment. For the detection of p34cdc~in anti-CYCl immunoprecipitates, the immunocomplexes were run on 0.1% SDS-12% polyacrylamide gels, electroblotted onto nitrocellulose filters and reacted with the PSTAIR-specific mAb. The histone Hl PK reaction was determined as follows: the anti-CYCl immunocomplexes were washed three times in kinase buffer (50mM TrisHCI pH 7.4/20mM MgCl,/l mM DTT/lOmM EGTA) and resuspended in 40 ul of kinase buffer supplemented with 50 @g/ml of histone HI (Boehringer Mannheim)/IO uM ATPjl mM CAMPdependent PK inhibitor peptide (P3294, Sigma). Reactions were initiated by the addition of 5 uCi of [7-3zP]ATP (3000 Ci/mmoI, Amersham), incubated for 30 min at 30°C and stopped by the addition of one volume of 2 x Laemmli’s sample buffer. The reaction products were analyzed on a 0.1% SDS-15% polyacrylamide gels. Only one fifth volume of the total reaction was loaded on the gel.
different organisms the same phase of the cell cycle may be controlled by CYCs belonging to different sequence classes. (2) Expression of CYCl in fission yeast rescued a cdcl3’” mutation indicating that the trypanosome homologue is capable of functioning as a mitotic regulator in fission yeast. This result strongly suggests that CYCl participates in the control of trypanosome mitosis. (3) In trypanosome cells CYCl associates with a 34-
kDa polypeptide antigenically related to ~34’~‘~and the resulting complex can catalyze the phosphorylation of histone H 1. (4) The abundance of CYCl is periodic with respect to the parasite cell cycle. When T. brucei cells were synchronized by hydroxyurea treatment and CYCl expression monitored by Western blotting of different time point samples taken after the arrest, it was found that CYCl accumulates late in the cell cycle, near the time of mitosis.
81
I abcdefg
s
%
2
C
-20
0. 0
1 -10
0
0
I
12
I
I
3
hours Fig. 6. Expression
of CYCI
during
I
4
I
5
after
I
I
6
I
78
I
9
cyc1
0 10
actin abcdefg
release
T. brucei cell cycle. (A)T. brucei cells were synchronized
by treatment
with 25 ug/ml
of hydroxyurea
for 9 h.
[sH]thymidine incorporation into DNA and the proportion of cells in late G2/M phase were determined in parallel samples of cells taken at different times after release from the arrest. (B) Protein lysates were prepared prior to (lane a) and at 1.5 h intervals after (lanes b-g) release from hydroxyurea treatment, and equal amounts of protein extract were Western blotted either with anti-CYCl serum (top) or the PSTAIR-specific mAb (bottom). (C) Total RNA (10 ug), prepared from cell samples taken at the time points indicated above, were analysed by Northern blotting using a CYCI specific probe (top). The blot was boiled for 10 min and rehybridized the GljS boundary by treatment with hydroxyurea (Steinert,
with an actin-encoding gene probe (bottom). Methods: Parasites were synchronized at 1969; Simpson and Braly, 1970). Procyclic forms were grown in Cunningham medium
to a cell number of lO’/ml followed by addition of 25 ug/ml of hydroxyurea and incubation for 9 h. Parasites were then harvested, washed three times in PBS and inoculated into fresh medium. Samples were taken prior to and at 1.5 h intervals following removal of the drug for preparation of protein
extracts
and
purification
of total
cellular
RNA.
To monitor
the progression
through
the cell cycle
of the trypanosome
population,
C3H]thymidine incorporation into DNA was determined every hour throughout the experiment. Samples of 2 x lo6 cells were pulsed for 15 min with 10 uCi of C3H]thymidine (48 Ci/mmol, Amersham) and radioactivity incorporated into trichloroacetic acid precipitable material was measured in a Beta counter. was determined
In addition,
the percentage
by microscopic
of cells having
examination
two kinetoplasts
of trypanosome
smears
and one nucleus,
stained
Our results also indicate that the periodic accumulation of CYCI gene transcript may contribute to the abundance of CYCl in G2. An interesting outcome of the studies described in this paper is the identification of a putative ~34’~‘~homologue in trypanosomes. An immunoreactive polypeptide of 34 kDa was evident when protein blots of T. brucei were reacted with the PSTAIR-specific mAb. This related protein was also detected in anti-CYCl immunoprecipitates which could catalyse the phosphorylation of histone HI, indicating that the trypanosome Cdc2 homologue binds to CYCs to form complexes displaying PK activity. Therefore, trypanosomes seem to conform to the emerging universal model in which Cdc2 requires its association with a CYC protein for its function in the cell cycle control.
ACKNOWLEDGEMENTS
We are grateful to Dr. Masakane Yamashita for providing the PSTAIR mAb and to Dr. David Beach for the generous gift of the Sz. pombe mutant strain and the
indicative
of late G2/M
phase (Diffley and Mama,
1989),
with Giemsa.
pAALN vector. We thank Maurice Steinert for advice on synchronization of T. brucei cultures and Marc Colet for the expert advice on computer based analysis of protein sequences. This work was supported by funds from the Commission of the European Communities (CI 1 900656 to J.L.A).
REFERENCES Affranchino, Macina,
J.L., Ibafiez, R., Aslund,
CL, Luquetti, L., Pettersson,
A., Rassi, U. and
A, Reyes, M.B., Frasch, A.C.C.:
Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas disease. Mol. Biochem. Parasitol. 34 (1989) 221-228. Affranchino, J.L., Pollevick, G. and Frasch, A.C.C.: The expression of a major shed Trypanosoma cruzi antigen results from the developmentally-regulated transcription of a small gene family. FEBS Lett. 280 (1991) 316-320. Alexandre, S., Paindavoine, P., Tebabi, P., Pays, A., Halleux, S., Steinert, M. and Pays, E.: Differential expression of a family of putative adenylate/guanylate cyclase genes in T. brucei. Mol. Biochem. Parasitol. 43 (1990) 279-288. Auffray, C. and Rougeon, F.: Purification of mouse immunoglobulin heavy chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107 (1980) 303-314.
82 Booher, R. and Beach, D.: Involvement of cdcl3’ in mitotic control in Schizosaccharomyces pombe: possible interaction of the gene product with microtubules. EMBO J. 7 (1988) 2321-2337. Boothroyd, J.C. and Cross, G.A.M.: Transcripts coding for variant surface glycoproteins have a short identical exon at their 5’ end. Gene 20 (1982) 281-289. Cunningham, I.: New culture medium for maintenance of tsetse tissues and growth of Trypanosomatids. J. Protozool. 24 (1977) 325-329. Dayhoff, M.O., Barker, W.C. and Hunt, L.T.: Establishing homologies in protein sequences. Methods Enzymol. 91 (1983) 524-545 Devereux, J.. Haeberli, P. and Smithies, 0.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 3877395. Diffley, P. and Mama, K.: Fixed and temporary Auctuations in the cell cycle of monomorphic lines of T. brucei gambiense. Mol. Biochem. Parasitol. 32 (1989) l-6. Draettd, G., Luca. F., Westendorf, J., Brizuela, L., Ruderman, J. and Beach, D.: cdc2 protein kinase is complexed with both cyclin A and B: evidence of proteolytic inactivation of MPF. Cell 56 (1989) 829-838. Dunphy, W.G. and Kumagai, A.: The cdc25 protein contains an intrinsic phosphatase activity. Cell 67 (1991) 1899196. Gautier, J., Minshull, J., Lohka, M., Glotzer, M., Hunt, T. and Maller, J.L.: Cyclin is a component of maturation-promoting factor from Xenopus. Cell 60 (1990) 487-494. Gautier, J.. Solomon, M.J., Booher, R.N., Bazan, J.F. and Kirschner, M.W.: cdc25 is a specific tyrosine phosphatase that directly activates p34C”CZ.Cell 67 (1991) 197-211. Ghiara, J.B., Richardson, H.E., Sugimoto, K., Henze, M.. Lew, D.J., Wittenberg, C. and Reed, S.I.: A cyclin B homolog in Saccharomyces cereuisiae: chronic activation of the cdc28 protein kinase by cyclin prevents exit from mitosis. Cell 65 (1991) 163--174. Glotzer, M., Murray, A.W. and Kirschner, M.W.: Cyclin is degraded by the ubiquitin pathway. Nature 349 (1991) 132-138. Gubler, U. and Hoffman, B.J.: A simple and very efficient method for generating cDNA libraries. Gene 25 (1983) 263-269. Hadwiger, J.A., Wittenberg, C., Richardson, H.E., Barros Lopes, M. and Reed, S.I.: A family of cyclin homologs that control the Gl phase in yeast. Proc. Natl. Acad. Sci. USA 86 (1989) 6255-6259. Hagan, I., Hayles, J. and Nurse, P.: Cloning and sequencing of the cyclin related cdcl3’ gene and a cytological study of its role in fission yeast mitosis. J. Cell Sci. 91 (1988) 587-595. Henikoff, S.: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28 (t 984) 35 l-359. Laemmli, U.K.: Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. Lehner, C.F. and G’Farrell, P.H.: The roles of Drosophila cyclins A and B in mitotic control. Cell 61 (1990) 533-547. Lew, D.J., Dulic, V. and Reed, S.I.: Isolation of three novel human cyclins by rescue of Gl cyclin (cm) function in yeast. Cell 66 (1991) 1197-1206. Minshull, J., Golsteyn, R., Hill, C.S. and Hunt, T.: The A- and B-type
cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBO J. 9 (1990) 2865-2875. Moreno, S. and Nurse, P.: Substrates for p34’% in vivo veritas? Cell 6i (1990) 549-551. Moreno, S., Klar, A. and Nurse, P.: Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194 (1991) 795-823. Nurse, P. and Bisset, Y.: Gene required in Gl for commitment to the cell cycle and G2 control of mitosis in fission yeast. Nature 292 (1981) 558-560. Pines, J. and Hunt, T.: Molecular cloning and characterization of the mRNA for cyclin from sea urchin eggs. EMBO J. 6 (1987) 2987-2995. Pines, J. and Hunter, T.: Isolation of human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdcz. Cell 58 (1989) 833-846. Revelard, P. and Pays, E.: Structure and transcription of a P-ATPase gene from ~r~pa~oso~a brucei. Mol. Biochem. Parasitol. 46 (1991) 241-252. Saiki, R.K., Schaf, S.. Faloona. F.. Mullis, K.B., Horn, G.T., Ehrlich, H.A. and Arnhein. N.: Enzymatic amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230 (1985) 1350-l 354. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with the chain-termination inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Simpson, L. and Braly. P.: Synchronization of Lei~hman~u tarentolae by hydroxyurea. J. Protozool. 17 (1970) 511-517. Smith, D.E. and Fisher, P.A.: Identification, developmental regulation and response to heat of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J. Cell Biol. 99 (1984) 20-28. Steiner& M.: Reversible inhibition of the division of Crith~d~a lu~jl~ue by hydroxyurea and its use for obtaining synchronized cultures. FEBS Lett. 5 (1969) 291-294. Swenson, K.I., Farrell, K.M. and Ruderman, J.V.: The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes. Cell 47 (1986) 861-870. Thomas, P.S.: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77 (1980) 5201-5205. Wang, J., Chenivesse, X., Heuglein, B. and Brechot, C.: Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma. Nature 343 (1990) 555-557. Xiong, Y. and Beach, D.: Population explosion in the cyclin family. Curr. Opinion Biol. 1 (1991) 362-364. Xu, H.P., Rajavashisth, T.. Grewal, N., Jung, V., Riggs, M., Rodgers, L. and Wigler, M.: A gene encoding a protein with seven zinc finger domains acts on the sexual differentiation pathways of Schizosaccharomyces pombe. Mol. Biol. Cell 3 (1992) 721-734.
GENE
B.V. All rights reserved.
0378-l 119/93/$06.00
0729 1
Isolation of a mitotic-like cyclin homologue from the protozoan Trypanosoma brucei (Cell cycle; cDNA cloning; polymerase chain reaction; c&2; M-phase kinase; mitosis; destruction box)
JosC L. Affranchino, Silvia A. Gonzalez and Etienne Pays Department of Molecular
Biology, University of Brussels, 1640, Rhode St. Get&e. Belgium
Received by J.R. Kinghorn:
24 November
1992; Revised/Accepted:
5 April/l5
April 1993; Received at publishers:
27 May 1993
SUMMARY
Activation of the p34cdc2protein kinase (PK) at different stages of the eukaryotic cell cycle is controlled by interaction with regulatory proteins known as cyclins (CYCs). Using a probe obtained by PCR amplification, we have isolated from the protozoan, Trypanosoma brucei, a cDNA clone encoding a CYC homologue. The amino acid sequence deduced for this gene (CYCI) shares structural homology with A- and B-type CYCs of other organisms, including a motif, the destruction box, which has been related to the rapid turnover of these CYC proteins in mitosis. When expressed in fission yeast, CYCZ is able to rescue the defect of a temperature-sensitive cdcl3 mutant, demonstrating that it is functional as a cell-cycle regulator. In trypanosome cells, CYCl associates with a 34-kDa protein that cross-reacts with a monoclonal antibody against the conserved ‘PSTAIR’ epitope of p34cdc2, and the complex displays histone Hl PK activity. Furthermore, when trypanosome cells are synchronized by hydroxyurea treatment, CYCl accumulates as cells progress towards mitosis. These observations, taken together, suggest that CYCl is a component of the active PK complex required for the control of trypanosome mitosis.
INTRODUCTION
The eukaryotic cell cycle is controlled at two major decision points, the transitions GljS and G2/M, by the activity of a 34-kDa serine-threonine PK which is the product of the cdc2 gene (Nurse and Bisset, 1981). It is Correspondence to: Dr. J.L. Affranchino at his present address: Centro de Virologia Animal (CEVAN), Serrano 661 (1414), Buenos Aires, Argentina. Tel. (54-l) 857 0012; Fax (54-l) 856 4495. Abbreviations: aa, amino acid(s); bp, base pair(s), BSA, bovine serum albumin; cDNA, DNA complementary to RNA; CYC, cyclin; CYCI, gene encoding CYCl; DMSO, dimethylsulfoxide; kb, kilobase or 1000 bp; mAb, monoclonal antibody(ies); nt, nucleotide(s); ORF, open reading frame: oligo, oligodeoxyribonucleotide; PBS, phosphatebuffered saline (0.137 M NaC1/0.0027 M KCl/0.0043 M Na,HP0,/0.0014 M KH,PO,); PCR, polymerase chain reaction; PK, protein kinase; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/O.OlS M Nasecitrate pH 7.6; Sz.. Schizosaccharomyces; T., Trypanosoma; ts, temperature sensitive.
thought that the p34’*” PK phosphorylates a variety of substrates that are directly involved in the commitment of the cell to the events of chromosomal replication and mitosis (Moreno and Nurse, 1990). The activity of p34 PK depends on the dephosphorylation of Tyr and Thr residues by a phosphatase encoded by the cdc25 gene (Gautier et al., 1991; Dunphy and Kumagai, 1991). In addition to these post-translational modifications, regulatory proteins known as CYCs, that associate with ~34, confer kinase activity to the resulting complex (Draetta et al., 1989; Gautier et al., 1990). CYCs can be classified into two major groups on the basis of their accumulation and functional role during specific phases of the cell cycle and the presence of conserved motifs within their aa sequence. One group includes mitotic CYCs, consisting of A and B classes, that exhibit maximal levels at the G2/M transition and rapidly decrease during M phase. This abrupt decline allows cells to complete mitosis. They have been described in a vari-
76
ety of organisms including yeast (Hagan et al., 1988; Booher and Beach, 1988; Ghiara et al., 1991) sea urchin (Pines and Hunt, 1987), frog (Minshull et al., 1990), fly {Lehner and O’Farrell, 1990), and man (Fines and Hunter, 1989). The second group corresponds to CYCs that are required by the cells for passage through GljS transition to undergo DNA replication. They accumulate during Gl and are degraded in S phase (Xiong and Beach, 199 1). Although extensive information has been gathered on yeast, invertebrates and vertebrates, nothing is known about cell cycle regulation in Protozoa. Trypanosomes are flagellated parasites which alternate between an insect vector and a mammalian host. Their life cycle shows transformations from dividing into non-dividing forms, offering an attractive model for cell division control studies. Proliferating forms are essential for the development of an infection, and differentiation to non-dividing forms is necessary for transmission between hosts. Therefore, knowledge of the genes and proteins involved in these processes might provide novel approaches to the control of infection and parasite proliferation in the human host. The aim of this paper was to isolate from Trypanosoma brucei a gene (CYCf) encoding a polypeptide structurally and functionally related to mitotic CYCs of other organisms.
RESULTS AND DISCUSSION
(a) Isolation of a cDNA clone encoding a T. brucei cyclin (CYC) homologue
A single degenerate oligo was designed utilizing the most conserved regions of published aa sequences of CYCs (Minshull et al., 1990) and considering the positional nt preferences of trypanosomes to limit the redundancy, and used as antisense primer in PCR amplifications. To devise the sense primer, we took advantage of the fact that trypanosomes bear the same 39-nt sequence (miniexon) at the 5’ end of their mRNAs (Boothroyd and Cross, 1982). When these primers were used in PCR experiments, a DNA fragment of 500 bp was consistently obtained from cDNA of procyclic forms of T. brucei. The amplified fragment was used in turn as radiolabelled probe to screen a h gt IO-cDNA library prepared from procyclic poly(A)‘RNA. This allowed the isolation of a cDNA clone (referred to as CYCI) which was sequenced. CYC’Zwas found to be a full-length gene since it extended from the miniexon sequence present at its 5’ end to a poly(A) tail at its 3’ end (Fig. 1). The isolated gene comprises an ORF of 334 aa which predicts a polypeptide of 36 27 1 Da (Fig. 1). The primary structure of the predicted ORF of CYCl
was found to have homology to previously characterized mitotic CYCs. CYCI contains a region of similarity to the cyclin box of A- and B-type CYCs, including sequence features expected to be conserved (Fig. 2). The alignment of CYCl with these CYCs in the cyclin-box region revealed 28% identity to sea urchin CYC B, 25% to human CYC Bl and fission yeast Cdcl3, and 26 % to both human and clam CYCs A (Fig. 2). For comparison, human CYCs A and B show 46% identity over the same region. If aa substitutions considered to be conservative are allowed, a higher degree of homology (46649%) is observed for all the comparisons, The degree of relatedness between mitotic CYCs and CYCl in the region shown in Fig. 2 is statistically significant as inferred from a statistical analysis based on the method of Dayhoff et al. (1983). Alignment scores of 5.6-5.8 standard deviations above the mean were obtained with respect to comparisons involving 100 randomly shuffled versions of the CYC 1 sequence. Although CYCl cannot be readily assigned to either A- or B-type, we assume that CYCl is most likely a mitotic CYC on the basis of the presence of a destruction box and its timing of expression during trypanosome cell cycle (see section e below). In CYCs A and B, the cyclin box is located after a long N-terminal domain. The aa sequence of the N terminus is highly variable, but a conserved motif involved in the programmed mitotic proteolysis of these proteins is embedded in this area (Glotzer et al., 1991). This region, called the destruction box, consists of the partially conserved sequence RXXLXXIXN followed by a Lys-rich region which provides potential sites for ubiquitination. Remarkably, CYCl, while lacking a divergent long N-segment (the homology with the cyclin box of mitotic CYCs starts at aa 60) shows in its C-terminal region a motif which clearly conforms to the destruction box (Fig. 1). The region corresponding to residues 255 263 (RXXVXXIXN) bears a single conservative aa substitution with respect to the consensus sequence and is followed by several scattered Lys residues (Fig. 1). Therefore, a destruction box which is a hallmark of mitotic CYCs, is also present in CYCl sequence, albeit located at the C-terminal segment. (b) Genomic organization
and transcription of CYCZ To examine the organization of CYCl in T. brucei
genome, a Southern blot analysis was performed. A radioIabelled probe spanning the complete ORF of CYCl was prepared and hybridized to restriction enzyme digestions of genomic DNA of procyclic forms of T. brucei. The results showed that CYCl is encoded by a single copy gene (Fig. 3A). The detection of two or three reacting bands in HindIII, PstI and Sal1 digests correlates with the presence of either one (Hind111 and &I) or two sites
77 GCGCACTGCATCTTGTAGCACGATGATGACAAACTTGAATTCCCC WMTNLNVRTIKGCFP
GGTTCCTTACCTGATGCACTCMCGACATGTCCGTCATCAG GSLPDALNDMSVINHILSLTGRFDEGQAAL
CTTCAGCACTTAGCGGAGGCACATATCATTTTACTACTACCGGAGGTMCACCCTTTTCG~ATACGACG~GGGAGG-CGGGCCTCGA~
LQHLAEAHllLLPEVTPFSLYDAGRKRASM CGTGTCTCCGGGCI\AGTTGCAGCTCGTGGTGTTGTCTTACCCC GQVAARGVVLVGGGCCFRAEHSDSANP R" s -CATGCGCACAGTACATGTTGTCGCGCGGG-~~GTGGATTCAGGGCGGCCGG~G-TGG*G~G~GGTGCG~GT~GAG K T CAQYHLSRENEVDSGRPVEMEEGGAKWE TACCTATGCGTTCACGGCCTCAGTGTGTTGCCGGGTATAT Y LCVHGLSVLPG~FCPQHSSRDATGLLLNE AGCTTTAGTAAGATGCTGACGGCATCCAACTGAGCGAGCGAGGGATTGGTGTCGACTGCAGGGCGGTG-CTG~GATGGGTGACGGTCGG SFSKMLKRHPTERGIGVDCRAVLLL~C TACCAGGTGCTGACGATAGCCAATAGAGAGAGAACTGT AN R E G Y 0" L T I
0
G
_R
RTASV~DINIQI~DVVEGN
GTGCAGACAACCACAATTCAC~CAGGGAAGCGTAGMG~~GC~CG-CCGTGTGGACCTGTCGTACGAGACCC~TTGAGGCG
v
PVVRDPFEA
OTTTIQQQGSVEELLRXPCG
TACTATGCRATGGCCMCCC~CTG~~MCAG~G~A~ATGTGCACCCA~TGATA~GG~GAGCCAG~~CGTTAGGCC Y Y A MAN PTALTEELLCAPR* CCGGTGTACGGACATTTTGGTAGCAGTGTCGTTTGAACGnGT TTGGAATCGACACATGTGCGCGCGTTCGTCAACATGTGAACTACAG (A)63
Fig. 1. Nucleotide sequence of CYCI cDNA and deduced aa sequence. The mini-exon sequence is boxed and the stop codon of the ORF is indicated with an asterisk. The cyclin destruction box and the Lys residues which follow this motif are underlined. Methods: The oligos used in PCR amplifications (Saiki et al., 1985) were as follows: sense primer, S-GTTTCTGTACTATATTG, corresponds to the short sequence of the mini-exon which is added to the 5’ end of all trypanosome mRNAs (Boothroyd and Cross, 1985); antisense primer, S-RGYVACDCCVACNA (R = A or G; Y = C or T; V =A or C or G; D= A or G or T; N= A or C or G or T) corresponding to aa LVGVT or LVGVA, was synthesized according to a highly conserved region of the cyclin box. cDNA was prepared from poly(A)+RNA of procyclic forms of T. brucei according to the method of Gubler and Hoffman (1983). cDNA (200 ng) was used as template in 100 pl reactions containing 10 mM Tri%HCl pH 8.3/50 mM KCl/l.5 mM MgClJ0.2 mM of each dNTPj5 pM of each primer/2.5 units of Taq polymerase (BRL). After an initial denaturing step of 5 min at 95°C amplification was allowed to proceed for 35 cycles (1 min at 95”C, 2 min at 38°C and 3 min at 72°C). The PCR product was gel purified and cloned into the SmaI site of pUCl9 plasmid vector. A radiolabelled probe made from the PCR clone was used to screen a hgtlO-cDNA library prepared from poly(A)+RNA of T. brucei procyclic forms. DNA from positive phage plaques was purified and the inserts subcloned into the EcoRI site of pUCl9. The nt sequence determination was performed on both strands by the dideoxynucleotide method (Sanger et al., 1977) using a modified T7 DNA polymerase (Pharmacia). Unidirectional deletions of the cloned DNA fragments were made with exonuclease III (Henikoff, 1984). At least three independent full-length cDNA clones were completely sequenced. DNA and protein sequences were analysed using computer programmes supplied by Genetic Computer Group, Inc. (GCG package version 7.1; Devereux et al., 1984). The nt sequence has been submitted to the EMBL database with the accession No. X65625.
(MI) for these restriction enzymes within CYCZ gene sequence. To assess the steady-state level of CYCl gene transcript in the proliferating stages of T. brucei life cycle, a Northern blot of poly(A)+RNA of procyclic and bloodstream forms was hybridized to the CYCZ probe. A single transcript of 1.4 kb was detected in both forms of the parasite (Fig. 3B). The size of the hybridizing RNA band is in good agreement with the length of the corresponding cDNA clone. (c) Trypanosome CYCI gene rescues fission yeast c&13’” mutation The structural similarity between CYCl and its counterpart in fission yeast suggests that these proteins might be functionally homologous. To examine whether the CYCI gene could provide the functions carried out by cdcl3, we determined if the trypanosome gene could
rescue a fission yeast cdcl3’” mutation. CYCI cDNA was subcloned into the pAALN vector and transformed into Schizosaccharomyces pombe ade6-210 cdcl3-117 leul-32 ts mutant. Leu+ transformants were selected either at the permissive (25°C) or the restrictive temperature (35°C). Fig. 4 shows that the CYCI gene could rescue the cdcl3 mutation. At least ten independent Leu+ transformants containing the CYCl gene were able to grow at the restrictive temperature (data not shown). These data establish that CYCl is able to function as a mitotic inducer in fission yeast and suggests that CYCl is likely to control mitosis in trypanosome cells. (d) CYCl associates to a CdcZ-related polypeptide To characterize the trypanosome CYCZ gene product, antiserum against the bacterially expressed protein was prepared, affinity purified and used to probe a Western blot of protein extracts of procyclic forms. The CYCI
kb
A
B
abcde
pb
I)
Fig. 2. Comparison of trypanosome CYCl with A- and B-type CYCs in the cyclin box region. (A) The protein encoded by CYCI (Tb) was aligned with human CYC A (HU A; Wang et al., 1990) and clam CYC A (Cl A, Swenson et al., 1986). The alignments of the three sequences are based on the outputs of pairwise alignments assisted by the BESTFIT program (Devereux et al., 1984). Boxes indicate identical residues or conserved aa substitutions (as assigned by the program BESTFIT) between CYCl and the other CYCs. Closed and open circles designate, respectively, the positions of identical and similar aa in CYCl sequence with respect to those most likely to be conserved in A-type CYCs. (B) Alignment of CYCl (Tb) with sea urchin CYC B (SU; Pines
- 1.4 kb
Fig. 3. Genomic organization and transcription of the trypanosome CYCI gene. (A) Genomic DNA of procyclic forms of T. brucei was digested
with BnmHI
(lane a), EcoRI (lane b), Hind111 (lane c), PstI
(lane d) and Sal1 (lane e), resolved on a 0.85% agarose gel and blotted onto a nitrocellulose membrane. The filter was hybridized to a CYCI radiolabelled probe. (B) Identification of a CYCl gene transcript. A Northern blot of poly(A)+RNA purified from bloodstream (b) and procyclic forms (p) of T. brucei was hybridized to a CYCI probe. Methods: Trypanosoma brucei bloodstream forms (AnTat 1.3A clone) and procyclic forms (AnTat l.lB clone) were used in these experiments. High molecular weight genomic DNA purification and Southern blotting
and Hunt, 1987), human CYC Bl (HU; Pines and Hunter, 1989) and fission yeast Cdcl3 (Booher and Beach, 1988; Hagan et al., 1988). Closed
were performed as previously described (Affmnchino et al., 1991). Total RNA was purified by the urea-LiCl method (Auffray and Rougeon, 1980). Poly(A)‘RNA (2 ug) was denatured by glyoxal-DMSO treatment
and open circles indicate, respectively, the positions of CYCl with identical and similar aa to those highly conserved in B-type CYCs.
(Thomas, 1980), resolved by electrophoresis and blotted onto nitrocellulose membranes
antiserum identified a polypeptide of an apparent molecular size of 36 kDa, which agrees with the predicted size calculated for the primary translation product of CYCI (Fig. 5A). Similar protein blots probed with the corresponding preimmune serum did not display any detectable signal (data not shown). To analyze whether a ~34’~” homologue exists in trypanosomes, a protein blot was probed with a mAb against the PSTAIR epitope present in all Cdc2 proteins described so far. An immunoreactive band of 34 kDa was clearly detected (Fig. 5A). This result prompted us to investigate whether CYCl associates with the identified Cdc2 homologue. An asynchronous culture of procyclic forms was metabolically labelled with [35S]methionine and parasite cell lysates were immunoprecipitated with CYC 1 antiserum. The anti-CYC 1 immunocomplexes showed, in addition to the CYCl band, a polypeptide with the expected electrophoretic mobility for the putative Cdc2 homologue (Fig. 5B).
on 1.5% (w/v) agarose gel (Thomas, 1980). All hybrid-
izations were carried out in 3 x SSCj5 x Denhardt’s solution/O. 1% (w/v) SDS/SO ug/ml salmon sperm DNA for 16 h at 65°C. Filters were washed three times at 65°C in 0.1 x SSC/O.l% SDS.
None of these bands were observed when immunoprecipitations were performed with preimmune serum (Fig. 5B). These results show that CYCl antiserum coimmunoprecipitates a protein of 34 kDa, presumably p34’d’2. To verify that this 34-kDa polypeptide was indeed the Cdc2 homologue, anti-CYCl immunoprecipitates were Western blotted and reacted with the PSTAIRspecific mAb. Fig. 5C shows that an immunoreactive band of 34 kDa was observed in CYCl immunocomplexes but could not be detected in immunoprecipitates prepared with preimmune serum. Taken together, these observations demonstrate the existence of a physical association between CYCl and a polypeptide antigenically related to ~34’~‘~. To determine whether these complexes exhibit PK activity, anti-CYCl immunoprecipitates were examined
79
Fig. 4. Rescue of the Sz. pombe cdcl3 ts mutation. Sz. pombe strain ade6-210 cdcJ3-117 Ieul-32 was transformed with the expression vector pAALN (c), the plasmid pAALN carrying the CYCZ cDNA in the correct transcription orientation (b) and pAALN containing CYCl in the reverse transcription orientation (d). Leu+ transformants (b-d) and the parental strain (a) were streaked on minimal medium plates and incubated either at the permissive (25°C) or restrictive temperature (35°C). Cells were allowed to recover for 24 h at 25°C before shifting to the restrictive temperature. The plates were photographed after 5 days of incubation. Methods: The complete trypanosome CYCI cDNA was cloned in the correct transcription orientation into the EarnHI site of the pAALN vector (Xu et al., 1992) containing the LEU2 gene as selectable marker. The plasmid was transformed into Sz. pombe ade6-210 cdcl3-117 leul-32 ts strain (SP731) by the Li.ace.tate method (Moreno et al., 1991). Leu+ transformants were isolated at 25°C by plating on Edinburgh minimal medium plates (Moreno et al., 1991) supplemented with adenine. As controls, cells were transformed with the pAALN vector alone and a plasmid containing the CYCl gene in the reverse transcription orientation.
for their ability to phosphorylate histone H 1. As expected, the immunocomplexes obtained with serum against CYCl displayed a specific histone Hl PK activity, while the control immunocomplexes (preimmune serum) failed to catalyze the phosphorylation of this substrate (Fig. 5D). Therefore, CYCl seems to exert its biological function by integration with ~34’~” into an enzymatically active PK complex. (e) CYCl levels are cell-cycle regulated A distinctive characteristic of CYCs is their oscillation through the cell cycle showing accumulation and destruction at defined points (Pines and Hunter, 1989). We therefore sought to determine whether the level of CYCl varies during the cell cycle. To this end, an exponentially growing culture of procyclic forms of T. brucei was synchronized by incubation with hydroxyurea for 9 h and released from the arrest by inoculation into fresh medium. Hydroxyurea treatment is the only method proved to be efficient for the synchronization of trypanosome cells 1969; Simpson (Steinert, and Braly, 1970). Synchronization was monitored by analysis of DNA synthesis and quantification of cells in late G2/M (Fig. 6A). Parallel samples were taken at different time intervals to prepare protein and RNA samples. Reaction of protein blots with CYCl antiserum and the PSTAIR-specific mAb allowed us to assess the protein levels of CYCl and p34’d” in each sample (Fig. 6B). The results showed that CYCl level began to rise after S phase completion peaking 6-7.5 h after release from the arrest (Fig. 6B). By contrast, ~34’~‘~was detected at constant levels throughout the experiment (Fig. 6B). To examine whether accu-
mulation of the CYCl gene transcript contributes to the regulation of CYCl polypeptide abundance, a Northern blot analysis was performed. Fig. 6C shows that the CYCl mRNA level increased as cells progressed towards mitosis, which correlates with the timing of accumulation of the corresponding protein. The pattern observed for the expression of CYCl suggests that it is required during GZ/M phase and that the availability of this CYC could be controlled at least in part by the regulated accumulation of the CYCZ gene transcript. (f) Conclusions We have isolated from T. brucei a gene encoding a polypeptide homologous to CYCs. The results presented herein constitute the first evidence for the existence of these cell cycle regulators in Protozoa. Four sets of evidence suggest that CYCl represents the trypanosome counterpart of the CYCs of other organisms: (I) The primary structure of CYCl shows significant similarity, indicative of structural homology, to mitotic CYCs. CYCl is less closely related to A- and B-type CYCs than they are to each other, which suggests that CYCl may represent a novel mitotic type or a highly diverged version of A- and B- CYCs. Regarding the structural divergence of CYCl, distinctive features of trypanosome protein sequences, such as peptide insertions within the region of homology with proteins of other organisms, have been well documented (Alexandre et al., 1990; Revelard and Pays, 1991). Besides, the identification of distantly related Gl CYCs in different organisms, such as human CYC C (Lew et al., 1991) and yeast CLN proteins (Hadwiger et al., 1989), supports the notion that in
80
c P36
cyc1 -o-p34 -
P34
1
2
1
2
1
2
1
histone Hl
2
Fig. 5. CYCl polypeptide forms a complex with a p34Cdczhomologue that phosphorylates histone HI in vitro. (A) Characterization of anti-CYCl antibodies. Western blots of protein extracts prepared from procyclic forms of T. brucei were incubated with affinity-purified Ab against CYCl (lane I), and a PSTAIR-specific mAb (lane 2). The molecular mass markers (kDa) are indicated on the left margin. (B) CYCI antiserum co-immunoprecipitates a 34-kDa polypeptide. Lysates prepared from [3sS]methionine-1abell~ parasites were immunopre~ipitat~ with either preimmune serum (lane 1) or anti-CYCl serum (lane 2). Immunocomplexes were resolved on a 0.1% SDS-lo% polyacrylamide gel (Laemmli, 1970) and autoradiographed. (C) The 34-kDa polypeptide co-immunoprecipitated by anti-CYCl antibodies is antigenically related to ~34’~“. Parasites were lysed and immunoprecipitated with either preimmune serum (lane 1) or anti-CYCl serum (lane 2). Immunocomplexes were resolved on a 0.1% SDS-12% polyacrylamide gel, transferred onto nitro~1lulo~ filters and reacted with the PSTAIR-s~cific mAb. The portion of the blot containing IgG was removed. (D) AntiCYCl immunoprecipitates exhibit histone Hl PK activity. Immunocomplexes prepared either with preimmune serum (lane 1) or anti-CYCl serum (lane 2) were examined for their ability to catalyse the phosphorylation of histone Hl. Methods: To obtain an immunogen for anti-CYCl antibodies production, the complete ORF of CYCI cDNA (bp 46-1078) was amplified by PCR and cloned into the EcoRI site of pKK223-3 plasmid vector (Pharmacia). This construct allowed the expression of the CYCt gene from its own initiating ATG. The recombinant polypeptide was expressed in Escherichiu cofi (JM109 strain) and isolated by electroelution from a preparative 0.1% SDS-12% polyacrylamide gel. Specific Ab against CYCI were raised in white New Zealand rabbits. The antiserum against CYCl was affinity purified by adsorption to and elution from nitrocellulose strips containing bacterially expressed protein (Smith and Fisher, 1984). Antibodies were eluted from the nitrocellulose with 0.1 M glycine pH 2.4 and immediately neutralized with one tenth volume of 1 M TrisHCl pH 8.0. Immunoblotting was performed essentially as described in Affranchino et al. (1989). Protein lysates from procyctic cells were resolved on 0.1% SDS-12% ~lyacrylamide gels and blotted onto nitrocellulose membranes. Filters were incubated either with affinity-purified anti-CYCl Ab (diluted 1:lOO)or with the PSTAIR-specific mAb (200 ng/ml). Antigen-antibody reaction was developed by incubation with i251-labelled Protein A (> lo7 dpm/pg, 5 x lo5 cpm/ml; Amersham). Rabbit anti-mouse serum (400 ng/ml) was included in the incubation mixture in the case of the membrane reacted with the mAb. For immunoprecipitations procyclic forms grown to a cell density of lO’/ml were starved for 2 h in methionine-deficient Cunningham medium (Cunningh~, 1977) and metabolically labelled for 2 h with 100 uCi/ml [3sS]methionine (1120 Ci/mmol, ICN Biomedicals). Parasites were harvested, washed twice in cold PBS and lysed at 0°C with 300 ~1 Lysis buffer (50 mM TrisHCl pH 8.0/150 mM NaCl/l% [w/v] NP-40/10 mM NaF/lOO uM NaV0,/60 mM B-glycerophosphate/S mM EDTA/300 pM PMSFjlO ug/ml trasylol/5 pg/ml leupeptin/l kg/ml pepstatin). After 30 min at O”C, lysates were spun at 12000 x g for 10 min and the resulting supernatants subjected to imm~oprecipitation assays. Lysates (100 ul) were precleared by the addition of 30 pl of protein A-Sepharose CL4B beads (Pharmacia) 10% (w/v) in lysis buffer + 1% (w/v) BSA. Samples were rotated end-over-end for 30 min at 4°C and centrifuged 1 min at 12000 x g. The supernatants were incubated with 1 ul of anti-CYCl serum for 2-4 h at 4°C. As control, lysates were immunoprecipitated with preimmune rabbit serum. Subsequently, a 30 ul sample of protein A-Sepharose beads 10% (w/v) in lysis buffer was added and the mixture rotated end-over-end during 1 h at 4°C. Beads were then pelleted and washed three times in lysis buffer + 0.1% (w/v) BSA. The immunocomplexes were released by boiling in 60 ~1 of 1 x Laemmli’s sample buffer (Laemmli, 19’70),resolved on a 0.1% SDS-IO% polyacrylamide gel and autoradiographed after fluorographic treatment. For the detection of p34cdc~in anti-CYCl immunoprecipitates, the immunocomplexes were run on 0.1% SDS-12% polyacrylamide gels, electroblotted onto nitrocellulose filters and reacted with the PSTAIR-specific mAb. The histone Hl PK reaction was determined as follows: the anti-CYCl immunocomplexes were washed three times in kinase buffer (50mM TrisHCI pH 7.4/20mM MgCl,/l mM DTT/lOmM EGTA) and resuspended in 40 ul of kinase buffer supplemented with 50 @g/ml of histone HI (Boehringer Mannheim)/IO uM ATPjl mM CAMPdependent PK inhibitor peptide (P3294, Sigma). Reactions were initiated by the addition of 5 uCi of [7-3zP]ATP (3000 Ci/mmoI, Amersham), incubated for 30 min at 30°C and stopped by the addition of one volume of 2 x Laemmli’s sample buffer. The reaction products were analyzed on a 0.1% SDS-15% polyacrylamide gels. Only one fifth volume of the total reaction was loaded on the gel.
different organisms the same phase of the cell cycle may be controlled by CYCs belonging to different sequence classes. (2) Expression of CYCl in fission yeast rescued a cdcl3’” mutation indicating that the trypanosome homologue is capable of functioning as a mitotic regulator in fission yeast. This result strongly suggests that CYCl participates in the control of trypanosome mitosis. (3) In trypanosome cells CYCl associates with a 34-
kDa polypeptide antigenically related to ~34’~‘~and the resulting complex can catalyze the phosphorylation of histone H 1. (4) The abundance of CYCl is periodic with respect to the parasite cell cycle. When T. brucei cells were synchronized by hydroxyurea treatment and CYCl expression monitored by Western blotting of different time point samples taken after the arrest, it was found that CYCl accumulates late in the cell cycle, near the time of mitosis.
81
I abcdefg
s
%
2
C
-20
0. 0
1 -10
0
0
I
12
I
I
3
hours Fig. 6. Expression
of CYCI
during
I
4
I
5
after
I
I
6
I
78
I
9
cyc1
0 10
actin abcdefg
release
T. brucei cell cycle. (A)T. brucei cells were synchronized
by treatment
with 25 ug/ml
of hydroxyurea
for 9 h.
[sH]thymidine incorporation into DNA and the proportion of cells in late G2/M phase were determined in parallel samples of cells taken at different times after release from the arrest. (B) Protein lysates were prepared prior to (lane a) and at 1.5 h intervals after (lanes b-g) release from hydroxyurea treatment, and equal amounts of protein extract were Western blotted either with anti-CYCl serum (top) or the PSTAIR-specific mAb (bottom). (C) Total RNA (10 ug), prepared from cell samples taken at the time points indicated above, were analysed by Northern blotting using a CYCI specific probe (top). The blot was boiled for 10 min and rehybridized the GljS boundary by treatment with hydroxyurea (Steinert,
with an actin-encoding gene probe (bottom). Methods: Parasites were synchronized at 1969; Simpson and Braly, 1970). Procyclic forms were grown in Cunningham medium
to a cell number of lO’/ml followed by addition of 25 ug/ml of hydroxyurea and incubation for 9 h. Parasites were then harvested, washed three times in PBS and inoculated into fresh medium. Samples were taken prior to and at 1.5 h intervals following removal of the drug for preparation of protein
extracts
and
purification
of total
cellular
RNA.
To monitor
the progression
through
the cell cycle
of the trypanosome
population,
C3H]thymidine incorporation into DNA was determined every hour throughout the experiment. Samples of 2 x lo6 cells were pulsed for 15 min with 10 uCi of C3H]thymidine (48 Ci/mmol, Amersham) and radioactivity incorporated into trichloroacetic acid precipitable material was measured in a Beta counter. was determined
In addition,
the percentage
by microscopic
of cells having
examination
two kinetoplasts
of trypanosome
smears
and one nucleus,
stained
Our results also indicate that the periodic accumulation of CYCI gene transcript may contribute to the abundance of CYCl in G2. An interesting outcome of the studies described in this paper is the identification of a putative ~34’~‘~homologue in trypanosomes. An immunoreactive polypeptide of 34 kDa was evident when protein blots of T. brucei were reacted with the PSTAIR-specific mAb. This related protein was also detected in anti-CYCl immunoprecipitates which could catalyse the phosphorylation of histone HI, indicating that the trypanosome Cdc2 homologue binds to CYCs to form complexes displaying PK activity. Therefore, trypanosomes seem to conform to the emerging universal model in which Cdc2 requires its association with a CYC protein for its function in the cell cycle control.
ACKNOWLEDGEMENTS
We are grateful to Dr. Masakane Yamashita for providing the PSTAIR mAb and to Dr. David Beach for the generous gift of the Sz. pombe mutant strain and the
indicative
of late G2/M
phase (Diffley and Mama,
1989),
with Giemsa.
pAALN vector. We thank Maurice Steinert for advice on synchronization of T. brucei cultures and Marc Colet for the expert advice on computer based analysis of protein sequences. This work was supported by funds from the Commission of the European Communities (CI 1 900656 to J.L.A).
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