Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Involvement of CDK/cyclin motif in ciliate cell cycle regulation Zhang, Hong 2000

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2000-566536.pdf [ 11.07MB ]
Metadata
JSON: 831-1.0089702.json
JSON-LD: 831-1.0089702-ld.json
RDF/XML (Pretty): 831-1.0089702-rdf.xml
RDF/JSON: 831-1.0089702-rdf.json
Turtle: 831-1.0089702-turtle.txt
N-Triples: 831-1.0089702-rdf-ntriples.txt
Original Record: 831-1.0089702-source.json
Full Text
831-1.0089702-fulltext.txt
Citation
831-1.0089702.ris

Full Text

I N V O L V E M E N T OF C D K / C Y C L I N MOTIF IN CILIATE C E L L C Y C L E R E G U L A T I O N by HONG Z H A N G B. Sc., Fujian Normal University, China, 1987 M . Sc., Fujian Normal University, China, 1990 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF T H E REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Zoology, Faculty of Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A July 2000 © Hong Zhang, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^Z.&~&LOG, Y The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The eukaryotic cell cycle is controlled by oscillation of the activity of cyclin-dependent protein kinases (Cdks). Ciliates, with their elaborate cellular structures and unusual cell cycle organization, present interesting subjects for cell cycle studies. A novel Cdk termed PtCdk2 was isolated in Paramecium tetraurelia, and with PtCdkl and PtCdk3 forms the P. tetraurelia Cdk family. The lack of affinity of PtCdk2 for yeast pl3sucl protein distinguishes it from PtCdk3, the only Paramecium Cdk so far displaying pl3sucl binding. PtCdk2 displays a cell cycle-stage dependent histone HI kinase activity, peaking at the end of the vegetative cell cycle. Coincidence of the kinase activity peaks of PtCdkl, PtCdk2 and PtCdk3 with initiation of macronuclear D N A synthesis (IDS), cell division and point of commitment to cell division (PCD), respectively, suggests they play different roles in cell cycle control. Consistent with the conservation of cyclin-binding domains in the P. tetraurelia Cdks, two mitotic cyclin homologues, PtCycl and PtCyc2, have been identified. This is the first time that cyclin genes have been cloned in ciliates. Both PtCycl and PtCyc2 proteins show characteristic patterns of accumulation and destruction during the vegetative cell cycle, with PtCycl peaking at the PCD, and PtCyc2 reaching the maximal level at the end of the cell cycle. Results of coimmunoprecipitation experiments indicate that PtCycl and PtCyc2 are associated with PtCdk3 and PtCdk2, respectively. Study on the subcellular localization of TtCdkl in Tetrahymena thermophila by immunofluorescence microscopy reveals its association with the membrane-skeletal domains that surround mature but not nascent basal bodies in the cell cortex, suggesting that TtCdkl plays a role in the regulation of formation of the complex membrane-skeletal i i layer of this cell during division-related cortical morphogenesis. A partial TtCDKl knockout cell line constructed through somatic biolistic transformation resulted in a reduction of the regularity of the rows of basal bodies plus an additional effect on chromatin condensation in both macro- and micronuclei. M y work not only extends the ubiquity of Cdk/cyclin motif in the eukaryotic cell cycle regulation to ciliates, but also reveals interesting and unique roles of these molecules in cell cycle control in these organisms. TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS iv LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xiii ACKNOWLEDGMENTS xvi GENERAL INTRODUCTION AND BACKGROUND 1 A. Cyclin-dependent kinases - the master regulators of the eukaryotic cell cycle 2 B. Multiple Cdk/cyclin complexes regulate passage through the different stages of the cell cycle 7 C. A multitude regulatory mechanisms of Cdk activity 11 D. Cdks - keeping the cell cycle in order 23 E. Ciliate cell cycle 26 F. Rationale and research objectives 35 MATERIAL AND METHODS 38 A. Materials 38 B. Cell stock and Culture 38 C. Centrifugal Elutriation 39 D. Genomic D N A Preparation 40 E. R N A Preparation 40 iv F. Design of Oligonucleotide Primers and PCR Amplification 41 G. D N A library Screen 42 H . 3'and 5 'RACEs 42 I. Preparation of D N A Probes 43 J. Genomic Southern Blot Analysis 43 K. Protein Lysate Preparation 43 L. Generation of Polyclonal Antibodies Against Cdks and Cyclins 44 M . Western Blot Analysis 45 N . Immunoprecipitation 45 O. pl3™ c ; Binding Assay 46 P. Histone HI Kinase Assay 47 Q. Expression of GST Fusion Proteins in Bacteria 47 R. Indirect Immunofluorescence Microscopy 48 S. Targeted Disruption of the Macronuclear TtCDKl by Biolistic Transformation 48 T. Flow Cytometry Analysis 49 CHAPTER ONE. A NOVEL MEMBER OF THE CYCLIN-DEPENDENT KINASE FAMILY IN PARAMECIUM TETRAURELIA 51 1.1 Introduction 51 1.2 Results 52 v 1.2.1 Primary sequence predicts that PtCdk2 is a cyclin-dependent protein kinase 52 1.2.2 PtCdk2 exhibit extensive homology to Cdk homologues from other eukaryotes 57 1.2.3 The protein encoded by PtCDK2 corresponds to the polypeptide of 35 kDa recognized by anti-PSTAIRE antibody 60 1.2.4 p\3sucl binding property of PtCdk2 reveals a novel class of p34 c r f c 2 homologue in P. tetraurelia 61 1.2.5 PtCdk2 protein level does not vary during the Paramecium vegetative cell cycle 65 1.2.6 Cdk2 histone HI kinase activity peaks at the end of the cell cycle 65 1.3 Discussion 69 CHAPTER TWO. TWO DISTINCT CLASSES OF MITOTIC CYCLIN HOMOLOGUES, CYC1 AND CYC2, ARE INVOLVED IN C E L L C Y C L E REGULATION IN CILIATE PARAMECIUM TETRAURELIA 72 2.1 Introduction 72 2.2 Results 73 2.2.1 Identification of two distinct mitotic cyclin homologues from P. tetraurelia 73 2.2.2 Genomic organization of the P. tetraurelia cyclin genes 78 2.2.3 Paramecium mitotic cyclins do not belong to either A- or B-type cyclins 79 vi 2.2.4 PtCycl and Cyc2 protein levels display distinct cell cycle-dependent fluctuations in the vegetative cell cycle 80 2.2.5 PtCycl and PtCyc2 associate with different Cdks in P. tetraurelia 85 2.2.6 PtCycl and PtCyc2 form active histone HI kinases with respective Cdk partners 86 2.2.7 Conservation of cyclin-like sequences in ciliates 88 2.3 Discussion 92 C H A P T E R T H R E E . F U N C T I O N A L C H A R A C T E R I Z A T I O N OF A C Y C L I N -D E P E N D E N T P R O T E I N K I N A S E H O M O L O G U E , T t C d k l , IN A N O T H E R H O L O T R I C H O U S C I L I A T E TETRAHYMENA THERMOPHILA 96 3.1 Introduction 96 3.2 Results 97 3.2.1 TtCDKl encodes a homologue of cyclin-dependent kinases 97 3.2.2 TtCDKl is a member of multi-gene family of CDKs in T. thermophila.. .100 3.2.3 Periodic expression of T. thermophila Cdkl protein correlates with periodical activation of its histone HI kinase activity during the vegetative cell cycle 102 3.2.4 TtCdkl protein is spatially associated with basal body domains in the cell cortex 105 3.2.5 Functional characterization of TtCDKl gene by targeted gene knockout in somatic nuclei 107 3.3 Discussion 115 DISCUSSION A N D F U T U R E P R O S P E C T S 120 vn A. General Discussion 120 1. Multiple Cdk/cyclin complexes involved in the unicellular cell cycles of ciliates 122 2. Coordination of the cell cycle events occurred on the cell cortex and within nuclei through a cyclin-dependent protein kinase 123 3. Variations in the regulatory mechanisms for ciliate Cdks 125 B. Conclusions 127 C. Future Prospects 128 BIBLIOGRAPHY 131 APPENDICES 164 viii LIST OF TABLES Table 1. Mammalian Cdks and cyclins and their functions 5 Table 2. Cyclins in yeast 6 Table 3. Cdk inhibitors (CKIs) 20 Table 4. Percentage of amino acid identity between ciliate Cdks and Cdks from other eukaryotes 98 ix LIST OF FIGURES Fig. I - l . Activities and functions of various Cdk/cyclin complexes involved in the yeast and animal cell cycle controls 8 Fig. 1-2. Multiple modes of Cdk regulation 13 Fig. 1-3. A model ciliate cell showing functional differences between two types of nuclei 28 Fig. 1-4. Model for control of the vegetative cell cycles of P. tetraurelia (A) and T. thermophila (B) 32 Fig. 1-1. Nucleotide sequence of PtCDK2, and the predicted amino acid sequence of the gene product, PtCdk2 54 Fig. 1-2. Genomic Southern blot analysis of Paramecium CDK genes 56 Fig. 1-3. Comparison of the amino acid sequence of Cdk2 and Cdkl from P. tetraurelia with Cdk homologues from Trichomonas vaginalis, Entamoeba histolytica, Plasmodium falciparum, Dictyostelium discoideum, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Zea mays and Homo sapiens 58 Fig. 1-4. Detection of Cdk homologues in Paramecium lysate 62 Fig. 1-5. Specificity of the antibody against the Paramecium Cdkl peptide 62 Fig. 1-6. p l3 v " c 7 affinity of the Paramecium Cdks 64 Fig. 1-7. pl3'™c/ immunodepletion experiment showing PtCdk2 is a novel Cdk in P. tetraurelia 64 Fig. 1-8. Expression of PtCdk2 protein 67 Fig. 1-9. PtCdk2 exhibits protein kinase activity towards bovine histone HI which fluctuates in a cell cycle-dependent manner 68 Fig. 2-1. Genomic D N A sequences of P. tetraurelia CYCla (A) and CYC2 (B), and the predicted amino acid sequences of their gene products 76 Fig. 2-2. Genomic Southern blot analysis of Paramecium genomic D N A with probes derived from PtCYCla, CYClb, and CYC2, respectively 82 Fig. 2-3. Evolutionary tree of cyclins from P. tetraurelia (Pt), Spisula solidissima (Ss), Drosophila melanogaster (Dm), Xenopus laevis (XI), Homo sapiens (Hs), and Dictyostelium discoideum (Dd) 83 Fig. 2-4. Oscillation of PtCycl and PtCyc2 protein levels during the vegetative cell cycle of P. tetraurelia 84 Fig. 2-5. Western blot analysis of protein levels of PtCycl (A) and PtCyc2 (B) in P. tetraurelia cells that were exponentially growing or starved 84 Fig. 2-6. Interaction between Cdk and cyclin proteins in P. tetraurelia 87 Fig. 2-7. Oscillation of PtCycl and PtCyc2 associated histone HI kinase activity during the vegetative cell cycle of P. tetraurelia 89 Fig. 2-8. Alignment of cyclin-like sequences from B. intermedium, S. histriomuscorum, C. sp. and T. thermophila with corresponding cyclin box region of PtCycl A, B and PtCyc2 from P. tetraurelia 91 Fig. 3-1. Nucleotide sequence of TtCDKl, and the predicted amino acid sequence of the gene product, TtCdkl 99 Fig. 3-2. T. thermophila contains two anti-PSTAIRE-reactive polypeptides: p37 and p35 101 Fig. 3-3. T. thermophila Cdks exhibit different affinities for yeast pl3sucl protein 101 xi 3-4. TtCdk protein level and its histone HI kinase activity during the vegetative cell cycle 104 3-5. Localization of the TtCdkl protein in both wild-type and T t C D K I K O cells 108 3-6. Diagram of portions of the macronuclear wild-type TtCDKl gene (top) and the knockout construct (bottom) 112 3-7.Genomic Southern blot analysis to demonstrate partial disruption of the TtCDKl gene in T t C D K I K O cells 113 3-8. The TtCdkl protein level detected by western blot analysis in wild-type (WT) and T t C D K I K O cells grown in 50 p.g/ml (KO50) and 100 Ug/ml paromomycin (KO100) 113 3-9. Macronuclear and micronuclear D N A contents of both wild-type (WT) and T t C D K I K O (KO100) cells, as measured by flow cytometry 114 xii LIST O F ABBREVIATIONS microgram Hi microliter u M micromolar ATP adenosine triphosphate bp base pair(s) BSA bovine serum albumin C A K Cdk-activating kinase cAMP cyclic adenosine 3', 5' monophosphate Cdc/CDC cell division cycle Cdk cyclin dependent kinase cDNA complementary deoxyribonucleic acid CKI cyclin dependent kinase inhibitor DIG digoxigenin D N A deoxyribonucleic acid dNTP deoxyribonucleotide triphosphate DTT 1, 4 dithiothreitol E C L enhanced chemiluminescence E D T A ethyenediaminetetraacetate E G T A ethylene glycol-bis-N, N , N ' , N ' -tetraacetate FITC fluorescein isothiocyanate GSH glutathione GST glutathione S-transferase xiii h hour(s) HRP horseradish peroxidase IDS initiation of macronuclear D N A synthesis IgG immunoglobulin G IPTG isopropyl -D-thiogalactopyranoside kb kilobase kDa kiloDalton K L H keyhole limpet hemocyanin Mem mini-chromosome maintenance mg milligram min minute(s) ml millilitre MPF maturation-promoting factor/M phase promoting factor mRNA messenger ribonucleic acid MTOC microtubule organizing center ng nanogram nM nanomolar ORC origin recognition complex PBS phosphate buffer saline PCD point of commitment to cell division PCNA proliferating cell nuclear antigen PCR polymerase chain reaction P K A cAMP dependent protein kinase A xiv PMSF phenylmethyl sulfonyl fluoride pRb retinoblastoma protein R A C E rapid amplification of cDNA ends R N A ribonucleic acid SDS-PAGE SDS polyacrylamide gel electrophoresis ssrRNA small subunit ribosomal R N A TBS Tris buffered saline tris tris (hydroxymethyl) aminomethane U unit(s) X-gal 5-bromo-4-chloro-3-indolyl-|3-D-galactopyranoside Amino acids in protein sequences are indicated by the single letter code (Appendix IV). The ciliate Cdk and cyclin genes and their protein products have been named or renamed according to the newly proposed genetic nomenclature rules for ciliates (Allen et al'., 1998). Genes and their encoded proteins of other eukaryotic cells cited in the thesis have been written by following the conventional designations. X V ACKNOWLEDGEMENTS I am deeply indebted to my supervisor, Dr. Jim Berger, for the wonderful opportunity that he gave me to study in his laboratory, introducing me to the field of ciliate cell cycle, his invaluable guidance and persistent encouragement throughout my thesis work. I am grateful for the members of my supervisory committee, Drs. Hugh Brock, Tom Grigliatti, Don Moerman and Gerry Weeks, for many valuable suggestions and advice in every stage of my study. Special thanks to Dr. Tom Grigliatti and members of his laboratory for indispensable discussions and assistance in all these years. Dr. Gerry Weeks is also acknowledged for his critical reviewing of our manuscripts. I thank Dr. Martin Gorovsky for providing the plasmid p4T2-lAHindIII, Dr. Peter Bruns for sending me Tetrahymena strains, and Drs. Eric Meyer and James Forney for Paramecium genomic D N A library and cDNA library, respectively. M y thanks are also due to Dr. Elaine Humphrey for training with laser confocal microscopy and long hours of technical assistance, and Dr. David Ng for allowing the access of BioRad PDSIOOO/He particle delivery system. Many thanks to Dr. Liren Tang, not only for his pioneering work on the P. tetraurelia Cdks which made my work easier, but also for his tremendous help and wonderful friendship since the very first day of my study. Dr. Qianjing Zhang is thanked for his great assistance on flow cytometry analysis. My work was fund by the Natural Science and Engineering Council of Canada (NSERC) grant GOO-6300 to Dr. Jim Berger. xvi I owe a special debt to my wife, Xueqin, and my parents, both for their emotional support and forbearance throughout my years as a student. xvii GENERAL INTRODUCTION AND BACKGROUND The cell cycle is the period in the life of proliferating cells between one cell division and the next during which the events required for successful cell reproduction are completed. The eukaryotic cell cycle is divided into four stages: G l , S, G2 and M . G l is the gap phase during which cells prepare for the process of D N A replication. It is during the G l period that the cell integrates mitogenic and growth inhibitory signals and makes the decision to proceed, pause, or exit the cell cycle. An important checkpoint in G l has been identified in both yeast and mammalian cells. Referred to as 'START' in yeast and the 'restriction point' in mammalian cells, this is the point at which the cell becomes committed to D N A replication and completion of the cell cycle (Hartwell et al., 191 A; Nurse 1975; Pardee, 1974). S phase is defined as the period during which D N A synthesis occurs. The G2 interval is a second gap phase between the end of D N A replication and mitosis during which the cell prepares for the process of division. M stands for mitosis, the phase in which the replicated chromosomes are segregated into separate nuclei and cytokinesis occurs to form two independent daughter cells. In addition to G l , S, G2, and M , the term GO is used to describe cells that have exited the cell cycle and become quiescent. To duplicate, cells must generally double their contents, but must precisely solve two specific problems: they must replicate their D N A once and only once per cell cycle, and they must segregate their chromosomes precisely to daughter cells. In a normal cell cycle, S phase is always preceded by M phase and M phase does not begin until the previous S phase is completed (Nasmyth, 1993, 1996; Nurse, 1994). The cyclin-dependent kinases are key components of the processes that are responsible for the precise ordering and regulation of these events. 1 A. Cyclin-dependent kinases - the master regulators of the eukaryotic cell cycle. As soon as G l , S, G2 and M came to be thought of as major cell cycle states, identification of the factors that trigger the transitions between these states became one of the major goals of cell cycle research. The notion that a master regulator might orchestrate cell cycle progression stems primarily from cell fusion studies showing that the cytoplasm of M phase cells was capable of inducing nuclei from other stages to enter mitosis prematurely (Rao and Johnson, 1970). Maturation-promoting factor or M phase promoting factor (MPF) was first described in 1971 as an activity present in cytoplasm from metaphase-arrested frog eggs (Masui and Markert, 1971). Microinjection of MPF into G2-arrested frog oocytes causes them to enter meiotic metaphase without the need for protein synthesis. Such an activity was subsequently found in metaphase cells from almost all eukaryotes (Adlakha et ah, 1985; Gerhart et al., 1984; Hashimoto and Kishimoto, 1988; Kishimoto and Kanatani, 1976; Kishimoto et al., 1982; Weintraub et al., 1982) and was equally effective at promoting entry into M phase irrespective of the species of recipient or donor cells. This suggested that M P F was a universal regulator of the G2 to M phase transition in eukaryotic cells and probably contained evolutionarily conserved components. In the meantime, genetically tractable organisms, such as fungi, have provided a different avenue for the study of the mechanisms of the cell cycle progression. In Saccharomyces cerevisiae (Hartwell, 1974), Schizosaccharomyces pombe (Nurse et al., 1976), and Aspergillus nidulans (Morris, 1976), a variety of mutations that perturb the regulation of cell division have been identified and mutants have been isolated. For mitotic control in the fission yeast S. pombe, a network of interacting genes regulates the 2 onset of mitosis. A key component in this network is the p34c c protein kinase (Beach et al, 1982; Hindley and Phear, 1984; Nurse and Bisset, 1981; Simanis and Nurse, 1986), which is required for both the G2/M and Gl /S transitions of the cell cycle. The homologue of p34 c r f c 2 in S. cerevisiae (p34 C O C 2 S ) is also required for both 'START' and mitosis (Reed et al., 1985). Progress in the purification and characterization of M P F was delayed until a cell-free assay system from amphibian eggs was developed that could respond to added MPF by carrying out many of the processes that normally accompany mitosis such as nuclear disassembly and chromosome condensation (Lohka and Mausi, 1983; Lohka and Mailer, 1985, 1987; Miake-Lye and Kirschner, 1985). Using this system, Lohka et al. (1988) purified Xenopus MPF to near homogeneity and showed that active fractions contained a 34 kDa serine/threonine protein kinase and a 45 kDa phosphoprotein. The 34 kDa subunit was subsequently identified as p34cdc2, a Xenopus homologue of the cdc2 gene product from S. pombe and the CDC28 gene of S. cerevisiae (Dunphy et al., 1988; Gautier et al., 1988). The convergence of yeast genetics, frog biochemistry and mammalian tissue culture led rapidly to the identification of a population of similar protein kinase homologues from a variety of species that were activated at specific stages of the cell cycle. Since these kinases are only active when complexed with unstable regulatory subunits which were later called cyclins due to their fluctuations in abundance during the cell cycle, this family of protein kinases was referred to as the cyclin-dependent protein kinases (Cdks). It is the sequential activation and inactivation of Cdks, through the periodic synthesis and destruction of cyclins, which provide the primary means of cell cycle 3 regulation. In yeast, cell cycle progression appears to be regulated predominantly by a single Cdk, p24cdc2(CDC28> in complex with different cyclin subunits (Table 2). In contrast, metazoans, have evolved to use multiple Cdk catalytic subunits in complex with specific cyclins to regulate different stages of their cell cycles. So far, at least nine Cdks have been identified in mammals, which are referred to as Cdks 1-9 (Arellano and Moreno, 1997), although only Cdk l , Cdk2, Cdk4, Cdk6 and Cdk7 are directly involved in the cell cycle regulation while the others function in regulating transcription, D N A repair, differentiation, and apoptosis (Johnson and Walker, 1999; Nigg, 1995; Table 1). Cdks are highly conserved through evolution, in both structure and function. They share 40-65% overall identity at the amino acid level, and they also show extensive similarity with other serine/threonine protein kinases within their catalytic domains (Hanks et al., 1988). In domain III of protein kinases, Cdks all share a sequence related to the canonical E G V P S T A I P v E I S L L K E motif ( 'PSTAIRE' motif) found in yeast p34cdc2(CDC28>, which has now become the hallmark of the Cdk family. Mutations in this region impair or abrogate binding to cyclins, and anti-PSTAIRE antibodies recognize Cdks only as monomers (Pines and Hunter, 1990), suggesting that the PSTAIRE motif directly interacts with cyclin. The striking functional homology among eukaryotic Cdks has been demonstrated by the fact that the human homologue of S. pombe cdc2 was cloned by complementation using a fission yeast cdc2 mutant and a cDNA expression library prepared from human cells, and that several mammalian Cdks can replace the corresponding yeast proteins (Elledge and Spottswood, 1991; Lee and Nurse, 1987; Ninomiya-Tsuji et al., 1991). 4 Table 1. Mammalian Cdks and cyclins and their functions Cyclin Associated Cdk Function References A CDK1, CDK2 S phase entry and transition Guard etal, 1991; Pagano etal, 1992. B1 ,B2 CDK1 G2 exit, mitosis Pines and Hunter, 1991; Gallant andNigg, 1992. C CDK8 Transcriptional regulation, G0- Rickert etal, 1996. S transition D1,D2, D3 CDK4, CDK6 GO-S transition Xiong etal, 1992. E CDK2 Gl -S transition Ohtsubo et al, 1995. F ? G2-M transition Bai et al, 1994. G l , G2 CDK5 D N A damage response Home et al, 1996; Bates et al, 1996. H CDK7 Cdk activation, transcriptional Fisher and Morgan, regulation, D N A repair 1994;Makela et al, 1994. I ? ? Nakamura et al, 1995. K ? Transcriptional regulation, Cdk Edwards et al, 1998. activation T1,T2 CDK9 Transcriptional regulation Wei et al, 1998 5 Table 2. Cyclins in yeast Cyclins Organisms and Types Cdk partners Functions Cln l S. cerevisiae, G l CDC28 START Cln2 S. cerevisiae, G l CDC28 START Cln3 S. cerevisiae, G l CDC28 START Clb5 S. cerevisiae, B-type CDC28 D N A replication Clb6 S. cerevisiae, B-type CDC28 D N A replication Clb3 5. cerevisiae, B-type CDC28 G2-M C I M S. cerevisiae, B-type CDC28 G2-M Clb l S. cerevisiae, B-type CDC28 G2-M Clb2 S. cerevisiae, B-type CDC28 G2-M cigl S. pombe, B-type Cdc2 G2-M cig2 S. pombe, B-type Cdc2 START cdcl3 S. pombe, B-type Cdc2 G2-M Adapted from Pines (1995). 6 B. Multiple Cdk/cyclin complexes regulate passage through the different stages of the cell cycle. 1 Passage through 'START' or the 'restriction point'. In S. cerevisiae, the timing of START is determined by the transcription of G l cyclins which include Clnl , Cln2 and Cln3. Early studies proposed that they were functionally redundant or at least had partially overlapping functions (Richardson et al., 1989). It is now clear that Cln3 is sufficient for activation of START specific transcription factors, while Clnl and Cln2 could have a more specific role in regulation of other START-related events such as initiation of budding, D N A replication and inhibition of Clb2 proteolysis (Dirick et al, 1995; Stuart and Wittenberg, 1995). This leads to the idea that passage through START requires that activity of different cyclin/Cdk complexes, each of which performs a subset of the events necessary for the cell to enter S phase (Fig. I-l A). In S. pombe, five cyclin-like proteins have been identified (Fisher and Nurse, 1995). In normal cycling cells, cig2 is the cyclin that regulates the START transition (Martin-Castellanos et al., 1996). In the absence of other B-type cyclins, a single mitotic cyclin, cdcl3, can control progression through the cell cycle (Fisher and Nurse, 1996) (Fig. I-IB). In mammalian cells, several Cdks and cyclins regulate the cell cycle. The restriction point seems to be under the control of E - and D-type cyclins (Sherr, 1993) (Fig. I-1C). Both cyclins are synthesized sequentially during the G l interval and are rate-limiting for entry into S phase. The D-cyclins form complexes with CDK4 and CDK6, and are regulated by extracellular signals such as growth factors, anti-mitogenic factors 7 A . Saccharomyces cerevisiae > U Gl 1 S G2 M START t Cln3/Cdc28 Clnl,2/Cdc28^^ Cln5,6/Cdc28 jClb3,4/Cdc28^ I \ • yClbl,2/Cdc2\ time Function G l transcription Budding DNA replication G2/M G2/M B. Schizosaccharomyces pombe Gil S G2 M START > O U Function DNA replication G2/M time C. Animal cells Gl 1 S G2 M Restriction point | CycD/Cdk4,6 > a ^ y c E / C d k ^ /CycA/Cdk2 \ CvcA/Cdkl \ ^ y c B / C d k l ^ Function Growth factor signalling DNA replication DNA replication G2/M G2/M time Figure I-l. Activities and functions of various Cdk/cyclin complexes involved in the yeast and animal cell cycle controls. 9 and by contact inhibition. The tumor suppressor pRb has been proposed as a major target of CyclinD/CDK4 complexes (Ewen et al., 1993; Kato et ah, 1993). It has been shown that pRb binds and regulates a large number of cellular proteins, including members of the E2F family of transcription factors. (Johnson and Schneider-Broussard, 1998). E2F factors regulate the expression of many genes that encode proteins involved in cell cycle progression and D N A synthesis, including cyclin E and A, CDK1, dihydrofolate reductase, thymidine kinase, and D N A polymerase a. The pRb phosphorylation by these kinases at, or near, the restriction point results in dissociation of pRb from E2F and the expression of the above mentioned E2F-regulated genes that mediate advance of the cell through S phase. 2. Initiation ofS phase and entry into mitosis. After passage through the START/restriction point, S phase cyclin/Cdk complexes phosphorylate and activate proteins necessary for D N A replication. In budding yeast, Clb5 and Clb6/Cdc28 complexes are involved in promoting D N A replication although, in the absence of both cyclins, Clbl-4 cyclins can replace this function (Schwob and Nasmyth, 1993). Transcription of CLB5 and CLB6 genes depends upon prior expression of CLN G l cyclins, linking activation of the S phase signal to the completion of START. CLB3 and CLB4 genes encode two B-type cyclins that are expressed from the beginning of S phase until late in mitosis (Fitch et al., 1992; Richardson et al., 1992) (Fig. I-1A). The kinase activity is postulated to be involved in D N A replication and early mitotic spindle assembly as well as in meiosis II (Dahmann and Futcher, 1995; Grandin and Reed, 1993). 10 In animal cells, CDK2 is considered to be a key protein in the onset of D N A replication (Fang and Newport, 1991). Cyclin E and Cyclin A sequentially activate Cdk2 to drive cells into S phase (Girard et al, 1991; Pagano et al., 1992; Zindy et al., 1992) (Fig. I-1C). Cyclin A gene presents a similar expression pattern to that of CLB3 and CLB4 genes. Neutralizing Cyclin A function by microinjection of antibodies blocks the cell cycle at the G l /S transition (Pagano et al, 1992). Moreover, Cyclin A / C D K 2 complexes colocalize with D N A replication sites in S phase nuclei, suggesting a possible direct role in replication origin regulation (Cardoso et al., 1993). When Cyclin A specific antibodies are injected during or after S phase, cells complete D N A synthesis and are unable to initiate mitosis and arrest in G2. These data suggest a dual role for cyclin A in mammalian cells, regulation of D N A synthesis and contribution to the induction of mitosis (Minshull etal., 1990). The onset of M phase is regulated by a complex network of positive and negative activities organized in a cascade composed of protein kinases and phosphatases. CDK1 (p24cdc2<CDC28)) forms complexes with different mitotic cyclins (Cyclin A and B in higher eukaryotes, cdcl3 in fission yeast and Clbl-2 in budding yeast) (Fig. I-l). In most species, Cyclin B/CDK1 is maintained in an inhibited state during interphase and rises dramatically at the G2/M transition. The activation of this kinase is necessary but not sufficient for the completion of mitosis. Destruction of these mitotic complexes is necessary for exit from mitosis. C. A multitude of regulatory mechanisms of Cdk activity. As they play such a critical role in triggering cell cycle events Cdks are extensively regulated, perhaps more so than any other class of protein kinases. The small 11 Cdk catalytic subunit, which contains little more than the conserved catalytic core found in all eukaryotic protein kinases, lies at the heart of a network of regulatory pathways. Several different mechanisms are employed to regulate Cdk activity in order to ensure that the cell cycle is tightly controlled and yet remains exquisitely sensitive to changes in the environment. Cellular Cdk levels tend to remain constant during the cell cycle and are always in excess over cyclin (Draetta and Beach, 1989), and regulation of catalytic activity is primarily post-translational (Fig. 1-2). The typical Cdk catalytic subunit contains a 300 amino acid catalytic core that is completely inactive when monomeric and unphosphorylated. Cdks require binding of cyclins as an initial step in their activation process (Pines, 1995). Complete activation requires phosphorylation of the Cdk subunit, by the Cdk-activating kinase (CAK), at a conserved threonine residue (Thrl60 in human CDK2 and Thrl61 in human CDK1) (Kaldis et al, 1996; Thuret et al, 1996). Many Cdk-cyclin complexes can be further down-regulated by at least two major mechanisms: the phosphorylation of the Cdk subunit on inhibitory sites (Ffaese et al, 1995), and the binding of protein inhibitors known as CKIs (Cdk inhibitors) (Serrano et al, 1993; Toyoshima and Hunter, 1994). Targeted destruction of cell cycle effectors through the ubiquitin degradation pathway also plays a crucial role that can be either positive or negative depending on whether the protein being degraded is a cyclin or a CKI (Hershko, 1997). 12 Cyclin degradation Cyclin synthesis Thr14 phosphorylation (MYT1) Tyr15 phosphorylation (WEE1) Thr14/Tyr 15 dephosphorylation (CDC25) CKI binding Thr161 dephosphorylation Complex assembly S u c 1 b i n d j n g p h J ^ . ^ (CAK) Figure 1-2. Multiple modes of Cdk regulation. The human CDK1 is used as a reference for sites of phosphorylation. The names of enzymes responsible for phosphorylation/dephosphorylation are given in parenthesis. Arrows represent positive regulatory effectors of Cdk activity and 'T's indicate inhibition of Cdk activity. 13 1. Cyclin binding. Cyclins were originally identified by Tim Hunt on one-dimensional SDS-PAGE (SDS-polyacrylamide gel electrophoresis) as proteins in sea urchin eggs that were rapidly synthesized after fertilization (Pines, 1996). It is known that there was a striking change in the pattern of proteins synthesized before and after fertilization of marine invertebrate eggs (Rosenthal, et al., 1980). However, most of these proteins, such as the small subunit of ribonucleotide reductase (Standart et al., 1985), simply accumulated through the succeeding cell cycles. In contrast, cyclins were distinguished by their steady accumulation in interphase, followed by a specific and rapid proteolysis at mitosis (Evans et al., 1983). Clam Cyclin A was the first cyclin to be cloned and sequenced (Swenson et al., 1986). As more and more cyclins have been identified in a variety of organisms, cyclins are now more accurately defined as members of a family of structurally related proteins that bind and activate Cdk catalytic subunits. They are less conserved than Cdks. Homology among cyclins is typically limited to a relatively conserved domain of about 100 amino acids, the 'cyclin box' (Minshull et al., 1989; Pines and Hunter, 1989), which is responsible for Cdk binding and activation (Kabayashi et al., 1992; Lees and Harlow, 1993). Mutations in this region inhibit both binding and activation, suggesting that the two functions are not easily dissociated. Enzymatic measurements of the monomeric Cdk kinase activity versus the cyclin-bound forms have indicated that cyclin binding leads to a 40,000-fold increase in kinase activity (Connel-Crowley et al., 1993). The molecular basis of this dependence has now been revealed by the solution of the crystal structures of both an inactive monomeric CDK2 and an active CDK2/CyclinA complex (De Bondt et al., 1993; Jeffrey et al, 1995). From a comparison with the active 14 cAMP-dependent protein kinase A (PKA) structure (Knighton et al., 1991), two reasons for the inactivity of monomeric CDK2 were immediately apparent. Firstly, ATP was bound in a conformation that would preclude nucleophilic attack on the scissile P-y phosphate bond by the substrate hydroxyl group. Secondly, part of the C-terminal lobe of the kinase — the 'T-loop' domain — blocked the catalytic cleft. The main reason for the differences in structure between CDK2 and P K A was a cc-helical region (ccL12) unique to CDK2 that constrained both the ATP-binding site and the T-loop domain. Through the Cyclin A binding to the PSTAIRE region of CDK2 partners, the ccL12 helix is melted, which allows the ATP-binding site to reconfigure to resemble that of P K A and allows the T-loop to move away from the catalytic cleft. Each cyclin has a unique pattern of expression during the cell cycle. The timing of the expression of various cyclins is therefore key in determining at which phase of the cell cycle their associated Cdk is active. Consequently, cyclin abundance is rate limiting for progression through the different stages of the cell cycle. So far, 16 different cyclins have been described in mammals (Table 1), and at least nine distinct cyclins in S. cerevisiae and three in S. pombe are directly involved in cell cycle regulation (Table 2). Cyclins are frequently classified as G l cyclins or mitotic cyclins, depending on when in the cell cycle they function. Cyclin abundance is modulated at two levels, transcriptionally and by protein turnover. In mammalian cells, sequential oscillations in the levels of the major cyclins largely reflect mRNA levels. Cyclins are subject to proteolytic destruction by the ubiquitin-dependent proteosome pathway (Deshaies, 1995; Hochstrasser, 1995). The G l cyclins contain PEST sequences in the C-terminal regions that are thought to confer 15 instability, leading to a very short half-life (-20 min) (Rogers et al., 1986). Mitotic cyclins have a partially conserved sequence near the N-terminus called the 'destruction box'. Deletion or point mutations in the PEST sequence or in the destruction box block cyclin degradation, presumably by preventing ubiquitiation and subsequent proteolysis (Glotzer et al., 1991). Thus, it is easy to see how proteolytic destruction of cyclins provides an elegant way of making cell cycle progression an irreversible, one way process. Cyclin synthesis/destruction is an essential factor in determining the precise timing of activation of Cdks, however, phosphorylation adds a further critical step in the activation of these complexes. 2. Reversible phosphorylation. Multiple phosphorylation and dephosphorylation events occur on both Cdk and cyclin subunits. Whereas the functional significance of cyclin phosphorylation remains unclear and may be required for cyclin degradation (Won and Reed, 1996), the regulatory roles of Cdk phosphorylation are comparatively well understood. It is clear that phosphorylation controls the activity of Cdks both negatively and positively, depending on the phosphorylation sites. In the case of human CDK1, phosphorylation of Thrl4 and Tyrl5, two neighboring residues within the ATP binding domain, causes the CDKl /Cycl in B complexes to be inactive until the G2/M transition, when a dual specificity phosphatase, Cdc25, removes these phosphates and causes abrupt activation of the kinase (Galaktionov and Beach, 1991). In both S. pombe and vertebrates, but not in S. cerevisiae, the phosphorylation state of Tyrl5 depends on checkpoint controls that monitor D N A replication (Coleman andDunphy, 1994; Mailer, 1993). Genetic studies 16 first carried out in S. pombe have led to the identification of protein kinases (Weel/Mikl) as well as a phosphatase Cdc25 acting on Tyrl5. Interestingly, the Cdc25 and Weel proteins are phosphorylated by p34 c d c 2/cdcl3, generating a positive feedback loop (Fleig and Gould, 1991), which is important for the abrupt activation of the Cdk/cyclin complex at mitosis. Although bacterially expressed Cdks can form complexes with cyclins in the absence of any phosphorylation in vitro, they show barely detectable histone HI kinase activity (Solomon, 1993). This activity increases 100-fold upon phosphorylation of a highly conserved threonine residue (Thrl60 in human CDK2 and Thrl61 in human CDK1) in domain VIII of all members of the Cdk family (Solomon et al., 1992). Biochemical purification approaches were successful in identifying an abundant kinase that phosphorylates Thrl61, termed Cdk-activating kinase (CAK) (Fesquet et al., 1993; Poon et al., 1993; Solomon et al., 1993). Surprisingly, this kinase is itself a Cdk/cyclin complex that is present in the transcription factor TFIIH and able to phosphorylate the C-terminal domain (CTD) of the large subunit of R N A polymerase II (Feaver et al., 1994; Roy et al., 1994). This may indicate the existence of cross talk between the transcription machinery and cell cycle regulators mediated by this enzyme. In animal cells, the C A K complex has been demonstrated to include CDK7 and Cyclin H (Makela et al., 1994; Fisher and Morgan, 1994), and in fission yeast the homologous proteins are named mopl-mcs2 (Buck et al., 1995; Damagnez et al., 1995). A third component of the C A K complex, Mat l , was identified later, which is a 'RING' finger protein, and may function as an assembly factor that stabilizes interactions between the cyclin and the Cdk (Devault and Doree, 1995). The closest budding yeast homologue to 17 these kinases is Kin28, and seems to be involved only in TFIIH activity (Valay et al., 1993). A C A K functioning in vivo (Civl) has been reported recently, fully active as a monomer but devoid of CTD kinase activity (Thuret et al., 1996; Kaldis et al., 1996). These results suggest that budding yeast may have developed a different mechanism for p34 activation and raises the question of whether previous results concerning multimeric C A K s are relevant in vivo or only an in vitro phenomenon. Apart from some reduction in activity in quiescent cells, there is no evidence for variation in CDK7 activity during the cell cycle (Labbe et al., 1994; Tassan et al., 1994). Phosphorylation of Thrl60/161 is highly favored by cyclin binding, because binding causes a significant shift in this domain. The change in this region removes the blockage imposed by the T-loop on the catalytic cleft and further exposes the threonine residue, making it more accessible to C A K (Jeffrey et al., 1995). The recently solved structure of the CDK2/Cyclin A complex phosphorylated by the C A K on the Thrl60 has provided an interesting insight into the structural basis of Cdk activation by phosphorylation (Russo et al., 1996). The modified residue (phosphorylated Thrl60) nestles in a patch of basic residues on the C-terminal lobe of the kinase, anchoring the T-loop out of the way of incoming protein substrates and stabilizing the active site conformation. Thus, Thrl60 phosphorylation may not primarily affect catalytic activity but rather may affect the accessibility of the active site to protein substrates. 3. The role of Cdk inhibitors (CKIs). Inhibitors of Cdks and Cdk/cyclin complexes (CKIs) are the last players to join the cell cycle plot, but they have rapidly moved to center stage. They are usually small (15-27 kDa) proteins (Peter and Herskowitz, 1994). Most known CKIs are involved in 18 putting a brake on the cell cycle: some play a role in response to extracellular signals, whereas others appear to function in intrinsic steps of the cell cycle (Table 3). CKIs are able to associate in vivo with the Cdk subunit, the cyclin or the cyclin/CDK complex inhibiting their activity: inhibition of the Cdk complex kinase activity, interference with CAK-mediated Cdk activation or competition with cyclins for binding to the catalytic subunit. The inhibitory process can be carried out by one or by a combination of these mechanisms. The first CKIs were identified in yeast, where they function not only to mediate cell cycle arrest in response to antimitogenic factors but also to ensure that particular cell cycle events are initiated before others are completed. The former function is illustrated by FAR1 of S. cerevisiae: when FAR1 is activated (by phosphorylation) in response to the mating pheromone a-factor, it causes G l arrest via inhibition of p 3 4 C D C 2 S / C l n l and p34 C Z ) C 2 S/Cln2 (Peter etal, 1993; Tyers andFutcher, 1993). Sicl of S. cerevisiae exemplifies the latter function: Sic l inhibits p34 C D C 2 S /Clb5 and p34 C D C 2 S /Clb6 and thereby prevents D N A synthesis in G l phase. Following passage through START, it is degraded, thereby activating the Clb5 and Clb6 kinases, which are necessary for D N A replication (Donovan et al, 1994; Mendenhall, 1993; Schwob et al, 1994). A role in the correct ordering of cell cycle events has also been attributed to the ruml gene product of S. pombe: The ruml gene was isolated as a gene that prevented the uncoupling of replication from mitosis on a high-copy plasmid (Moreno and Nurse, 1994). Overexpression of ruml prevents cells from initiating mitosis, and causes repeated rounds of D N A replication. Conversely, cells lacking ruml seem to be unable to prevent 19 Table 3. Cdk inhibitors of yeast and mammalian cells Inhibitor Organism Target Cdk Physiological role References FAR1 S. cerevisiae CDC28 Pheromone response Peter & Herskowitz, 1994. SIC1 S. cerevisiae CDC28 Ordering the cell cycle events Schwobef a/., 1994. ruml S. pombe Cdc2 Ordering the cell cycle events Moreno & Nurse, 1994. p21 Mammals Multiple p53 responsive gene Xiong et al, 1993. CDKs pl6 Mammals CDK4/6 G l /S transition L i et al, 1994. pl5 Mammals CDK4/6 TGF-p response Hannon & Beach, 1994. p27 Mammals CDK2 TGF-p response/contact Polyakera/., 1994. inhibition 20 themselves from going into mitosis from the pre-START phase of G l . There are also indications that it may be able to target the cdcl3 for destruction (Hayles et al., 1994). CKIs also play multiple roles in vertebrates. Vertebrate CKIs described to date could be grouped in two families: the INK4 family, composed of pl5, pl6, pl8 and pl9, and the Cip/Kip family, including p21 c i p l , p27 k i p l and p57 k i p 2 . A hallmark of the INK4 family is their specificity for binding to CDK4 and CDK6. Inhibition of Cyclin D binding to CDK4 and CDK6 is likely to be the primary means of action of the INK4 family of inhibitors (Serrano et al., 1993). The gene for pl6 is a potential tumor suppressor gene. It is rearranged, deleted or mutated in a large number of tumor cell lines, and in some primary tumors, and it appears to play a unique role in regulating the status of pRb (Kamb et al., 1994; Nobori et al., 1994). In contrast, the Cip/Kip family inhibitors demonstrate a broad range of specificity and is able to inhibit all the G l cyclin/Cdk complexes and, to a lesser extent, CDKl/Cycl in B complexes (El-Deiry et al., 1993). Unlike the INK4s, this family has a higher affinity for the cyclin/CDK complex than for the monomeric C D K subunit; thus they inhibit the activity of the holoenzyme. p21 was identified almost simultaneously as a Cdk-binding protein (Harper et al., 1993; Xiong et al., 1993), a protein that is up-regulated in senescent cells (Noda et al., 1994), and a gene product that can be induced by the tumor suppressor p53 in response to D N A damage (El-Deiry et al., 1993). Induction of p21 inhibits cell cycle progression either by inhibiting a variety of cyclin/Cdk complexes or by inhibiting D N A synthesis through PCNA binding (proliferating cell nuclear antigen), an elongation factor for D N A polymerase 8. p27 has been implicated in mediating TGF-P as well as contact-induced inhibition of proliferation (Polyak et al., 1994). 21 4. Subcellular localization. Another mechanism employed by the cell to regulate Cdk activity is to localize them to particular subcellular compartments. Several studies have shown that some of these complexes may be regulated in part by their location in the cell, adding a further layer of complexity to the regulatory mechanisms. For example, Cyclin B l is translocated from the cytoplasm to the nucleus immediately prior to mitosis; the timing of this translocation may be a critical factor in determining the initiation of mitosis. Specific subcellular location has also been observed for a number of other cyclins, including Cyclins D, E, and B2 (Baldin et al, 1993; Jackman et al, 1995; Ohtsubo et al, 1995). By changes in localization, cyclins may act as a determinant of the substrates phosphorylated by Cdks. However, this issue needs further investigation. 5. The Cdk-interactingproteins - pl3sucl and its homologues. Another protein that interacts with, and may regulate p34 kinase activity is the product of the S. pombe sucl+ gene (pl3™ c 7). It was isolated as a high-copy suppressor of temperature-sensitive mutations of certain cdc2 alleles (Hayles et al, 1986). Like other fundamental regulatory proteins, pl3™ c ; homologues are found in divergent taxa from yeast to humans. A similar approach was used to isolate the S. cerevisiae sucl+ homologue, CKSl, which encodes an 18 kDa protein that is highly related at the amino acid level to the S. pombe pl3sucl. Interactions between p l3™ c ; / p l8 c * 5 ; and V2>4cdc2/CDC28 have been demonstrated in yeast by both genetic and biochemical approaches (Brizuela et al, 1987; Hayles, 1986; Hindley et al, 1987; Moreno et al, 1989). The extensive use of S. pombe p\3sucl affinity columns to isolate p34cdc2 kinase activity from a number of species also attests to a strong, conserved 22 interaction between two proteins (Arion et al, 1988; Dunphy and Newport, 1989; Hindley et al., 1987). In human cells, two homologues of the sucl+ and CKS1 genes, CKShsl and CKShs2, have been identified, and both are capable of rescuing a null mutation of the S. cerevisiae CKS1 gene when expressed from the S. cerevisiae GAL1 promoter (Richardson et al., 1990). p l3™ c ; / p l8 C A : s / is essential for cell viability in S. pombe (Hayles et al, 1986; Hindley et al, 1987; Moreno et al, 1989) and in 5. cerevisiae (Richardson et al, 1990). p l 3 i u c / is necessary for the inactivation of p34cdc2 at anaphase and for completion of mitosis in S. pombe. Cells of fission yeast that lack pl3sucl become locked in mitosis and arrest with elongated mitotic spindles that are characteristic of anaphase or telophase (Moreno et al, 1989). CKS1 deletion and analysis of temperature-sensitive CKS1 mutants in S. cerevisiae suggest that pl8CKS1 activity is required at both the Gl /S and G2/M transitions and affects bud formation (Hadwiger et al, 1989; Tang and Reed, 1993). Overexpression of pl3iUcI in S. pombe results in highly elongated cells with a G2 D N A content. Cells grow more slowly and show delayed entry into M-phase (Hayles et al, 1986; Hindley et al, 1987). Experiments in vitro using reconstitued Xenopus egg extracts suggest that excess pl3sucl may cause this delay by blocking Cdc25-dependent Tyrl5 dephosphorylation and subsequent kinase activation (Dunphy and Newport, 1988, 1989). However, pl3sucl binds to active p34cdc2 kinase suggesting that it does not inhibit catalytic activity in vitro. D. Cdks - keeping the cell cycle in order. In a normal cell cycle, D N A replication and mitosis are coupled and mutually dependent, thus ensuring their occurrence only once during each cell cycle. This 23 dependency between the two events appears to be maintained by checkpoint controls (Hartwell and Weinert, 1989) that act during the cell cycle to ensure that only one S phase takes place each cell cycle and that cells do not enter mitosis until D N A replication is complete. Together these checkpoints maintain the temporal order between S phase and mitosis, ensuring genome ploidy and integrity. Separate lines of investigations in different organisms, particularly yeast, frog and fly have demonstrated that these checkpoint controls act through Cdks. If D N A replication is not complete during S-phase then the onset of mitosis is blocked. This restraint is imposed through Tyrl5 phosphorylation by Cdc25. In fission yeast (Enoch et ah, 1991) and in Xenopus egg extracts (Smythe and Newport, 1992), it has been shown that arresting D N A replication inhibits the CDK1 (p34 c r f c 2) protein kinase by maintaining the inhibitory phosphorylation of Tyrl5. Mutants that have a reduced capacity to maintain Tyrl5 phosphorylation enter M phase even if S phase has been blocked. This view is consistent with the observation in mammalian cells that high level expression of CDK1 and Cyclin B induces entry into M phase even if S phase is not complete and that induction is prevented by simultaneous high level expression of the Weel protein kinase that phosphorylates Tyrl5 (Heald et al., 1993). It is now known in fission yeast that cells assess whether D N A replication is complete by monitoring if D N A replication complexes formed at initiation are present, as suggested by the study of mutants defective in or deleted for various genes encoding components of replication complexes including D N A polymerase a and initiation factors (D'Urso et al., 1995; Waseem et al., 1992). If they are present, then cells conclude that S-phase must be in progress, and so a signal is sent to block Tyrl5 dephosphorylation. A different 24 mechanism is applied by cells in G l which have not yet formed the replication complexes to inhibit the CDK1 protein kinase when cells are blocked in early G l before START (Moreno and Nurse, 1994). This acts through p25™"'7, a C D K inhibitor encoded by ruml. This protein is only present during the G l phase, and it inhibits activation of p34 c r f c 2/cdcl3 complex. The inhibition is due to p25™m/ directly inhibiting the protein kinase activity, and also because p25 r"m l promotes cdcl3 proteolysis. For the genome to remain stable it is important that there is only one S phase each cell cycle or ploidy will change. This control also involves Cdk kinases (Broek et al., 1991). A two-state model of initiation of D N A replication has been proposed to explain why eukaryotic cells replicate the genome once and only once per cell cycle (Nasmyth, 1996; Stern and Nurse, 1996). A detailed examination of the proteins associated with origins of replication in budding yeast has suggested that there are two different protein complexes at the origin - a pre-replicative complex which is competent for D N A replication, present during G l , and a post-replicative complex which is incompetent for D N A replication, present during the S, G2 and M phases (Li, 1995). In addition to ORC (origin-recognition complex) common to pre- and post-replicative complexes (Micklem et al., 1993; L i and Herskowitz, 1993), the former also includes some additional initiation proteins such as Cdcl8/cdc6 and Mem (mini-chromosome maintenance) family (Coleman et al., 1996; Kubota et al., 1995; Lopez-Girona et al., 1998). As a cell exits mitosis mitotic Cdk activity drops to a very low level and the initiation proteins become unphosphorylated, which leads to the initiation proteins binding to origins and the formation of pre-replicative complexes. Increase of the protein kinase to a moderate level at Gl /S allows pre-replicative complexes to be activated bringing about D N A replication. 25 In addition, it prevents any other pre-replicative complexes from being set up because phosphorylated initiation proteins cannot bind to origins. Maintenance of moderate protein kinase activity during G2 prevents reloading of the initiation proteins. Only after mitosis when protein kinase activity dropped could pre-replicative complexes be re-established. However, the dependency between D N A replication and mitosis can be disrupted during certain developmental processes: the dependency of mitosis upon D N A synthesis is broken in meiosis, where two successive events of chromosome segregation occur without an intervening D N A synthesis; and conversely, the dependency of D N A synthesis upon mitosis is broken in endoreduplication, where multiple rounds of D N A replication occur in the absence of intervening mitosis, leading to the production of chromosomes with doubling series of chromatids. During meiosis in clam and Xenopus oocytes, the mitotic cyclin Bs persist between the two-M phases, perhaps suppressing the intervening S phase (Kobayashi et al., 1991). In contrast, Drosophila salivary gland cells appear to lack mitotic cyclin B that could initiate M phase and, as a consequence, constantly redirect the cell back into G l , leading to repeated rounds of S phase (Lehner and O'Farrell, 1990). E . The Ciliate cell cycle. 1. The organisms. Ciliates are a group of diverse unicellular alveolate organisms that emerged prior to the appearance of the higher eukaryotes and are phyletically well separated from the eukaryotic lines that led to plants, animals and fungi. A phylogeny based on small subunit ribosomal RNAs (ssrRNAs) places ciliates among the 'crown group' of eukaryotes 26 (Knoll, 1992; Sogin, 1991). Ciliates are one of the three major groups within the alveolate superphylum that also includes the dinoflagellates and apicomplexans (Cavalier-Smith, 1993). In the 109 plus years since the evolutionary establishment of the first progenitor of the group, the ciliates have diverged into a rich assortment of subgroups containing many thousands of species. Despite their great genetic diversity, the ciliates remain united by two characteristics: the possession of complexes of cilia used for swimming or crawling and for phagocytic food capture, and the presence of nuclear dimorphism (Fig. 1-3). Although micro-and macronuclei develop from the mitotic products of a single zygote nucleus and reside in a common cytoplasm they differ from one another in almost every structural and functional characteristic (Karrer, 1986). The macronuclear D N A content may be several hundredfold that of the micronucleus. The polygenomic, somatic macronucleus is transcriptionally active and governs the phenotype in the vegetative cell cycle; the diploid, germline micronucleus is transcriptionally inert and responsible for maintaining genetic continuity. The macro- and micronucleus represent one of the simplest forms of soma and germline differentiation. Ciliates differ from metazoa mainly in that both germinal and somatic nuclei reside in the same cell throughout the life cycle. Two holotrichous ciliates, Paramecium tetraurelia and Tetrahymena thermophila, are used in this study. Though placed within a single major evolutionary lineage, the Oligohymenophorea, they are very different from each other as exemplified by their morphological characters and analysis of rRNA sequences. The evolutionary distance 27 Micronucleus Germ-line Diploid Transcriptionally inactive Mitotic Figure 1-3. A model ciliate cell showing functional differences between two types of nuclei. While both contain one macronucleus in each cell, P. tetraurelia has two micronuclei and T. thermpohila has only one. 28 between P. tetraurelia and T. thermophila based on ssrRNA sequences exceeds that between rat and brine shrimp (Frankel, 1999). P. tetraurelia ranges from 100 to 150 |im in length and 50 \im in maximum width. Each P. tetraurelia vegetative cell contains two micronuclei and one macronucleus. In bacterized phosphate-buffered wheat grass infusion the cell divides about every 6 h at 27°C. When a culture reaches stationary phase (>2,000 cells/ml), young clones undergo conjugation between two different mating types while older clones undergo autogamy (Berger and Rahemtullah, 1990; Sonneborn, 1959). At conjugation the micronuclei undergo meiosis and one meiotic product undergoes mitosis to produce a stationary and a migratory haploid nucleus (gametic nuclei). Reciprocal nuclear exchange and subsequent nuclear fusion then occur to produce a diploid zygotic nucleus (syncaryon) in each conjugant, which divides mitotically twice to produce progenitors for both new micronuclei and macronuclei while the old macronucleus degenerates. Autogamy is identical to conjugation except that the process occurs in a single cell, the migratory and stationary nuclei simply fuse to produce a zygotic nucleus. T. thermophila is about one third the size of Paramecium, and it has one macro-and one micronucleus in each cell. It can be grown in synthetic and chemically defined media under axenic conditions, with a doubling time of -2.5-3 h at 30°C. Since conjugation is the only sexual pathway in T. thermophila (i.e. no autogamy), a culture of high density of ~5xl0 5 cells/ml can be easily achieved in rich media. 2. Vegetative cell cycles of P. tetraurelia and T. thermophila. 29 In both Paramecium and Tetrahymena, macro- and micronuclei synthesize D N A at different times within the cell cycle, even though both types of nuclei are in the same cytoplasmic unit. Their D N A replication patterns are also regulated differently (Adl and Berger 1992; Frankel, 1999; Pasternak 1967; Rasmussen and Berger 1982; Rasmussen et al., 1986) (Fig. 1-4). In P. tetraurelia, macronuclear D N A synthesis begins at 0.25 of the cell cycle (Berger, 1971; Berger and Kimball, 1964; Rasmussen and Berger, 1982), and persists until the beginning of macronuclear elongation at about 0.9 in the cell cycle (Kaneda and Hanson 1974; Tucker et ah, 1980). There is no macronuclear G2 period. In contrast, micronuclear D N A synthesis is restricted to a very short period between approximately 0.35 and 0.65 in the cell cycle (Pasternak, 1967). The macronuclear S phase of T. thermophila occurs roughly in the middle of the cell cycle and takes about 1 h under optimal conditions (Frankel, 1999; McDonald, 1962). Micronuclear D N A synthesis in T. thermophila begins immediately after micronuclear division is completed and ends shortly after cytokinesis (McDonald, 1962); there is no G l period in the T. thermophila micronucleus. In both ciliates, the macronucleus divides by amitosis without notable chromosome condensation and spindle formation while micronucleus divides by a typical 'closed' mitosis without nuclear envelope breakdown. Given the imprecise nature of the amitotic division process, macronuclear D N A content is regulated through different but equally unorthodox mechanisms in P. tetraurelia and T. thermophila. An incremental mechanism is employed by P. tetraurelia whereby all cells synthesize a certain amount of D N A regardless of their initial D N A content, implying that the D N A content increases but does not double in cells with larger than average macronuclei, and increases more than two-fold in smaller than average 30 macronuclei. Because of this, half of the variance in D N A content introduced at each fission is removed during the course of the subsequent cell cycle (Berger 1979). In contrast, in T. thermophila, the increased variance in macronuclear D N A content introduced by amitosis is not removed within each round of D N A replication, which involves a complete doubling of D N A content (Andersen, 1977; Andersen and Zeuthen, 1971; Cleffmann, 1975; Doerder and DeBault, 1978). Instead, when macronuclear D N A content falls too low, two successive macronuclear D N A replications take place without an intervening cell division (Cleffmann, 1968). When macronuclear D N A content gets too high, two successive cell divisions take place without an intervening macronuclear S phase (Doerder and DeBault, 1978). 31 I D S I Gl I P C D C y t o k i n e s i s l A M l I M A C I Gl I S I M 1 M I C I 1 I I L 0 0.25 0.5 0.75 1.0 IDS P C D C y t o k i n e s i s 1 Gl s | AM | B 1 s 1 G2 1 M l S 1 Fig. 1-4. Model for control of the vegetative cell cycles of P. tetraurelia (A) and T. thermophila (B). The major control point of the cell cycle, the Point of Commitment to Division (PCD or physiological transition point/stabilization point), is shown with the cell cycle events of macronucleus (MAC) and micronucleus (MIC). The timing of the Initiation of Macronuclear D N A Synthesis (IDS) is determined at PCD in the preceding cell cycle in P. tetraurelia, but is set just prior to IDS in the same cell cycle in T. thermophila. 32 The major difference in the cell cycle control between P. tetraurelia and T. thermophila lies in the control of the initiation of macronuclear D N A synthesis (IDS). Nutrient down/up shift experiments in P. tetraurelia indicated that the temporal locations of the two major control functions, commitment to IDS and commitment to cell division (PCD) occur together around 90 min before cell division, rather than widely separated points as in typical eukaryotic cell cycles. At this point, P. tetraurelia cells become committed to division in the present cell cycle and the nature of the next cell cycle (meiotic or vegetative) is set, and the duration of the next G l interval is established (Berger, 1988). Therefore, the initial cell cycle control point in P. tetraurelia is actually located prior to fission during the preceding cell cycle, suggesting that from a regulative standpoint the cell cycles of P. tetraurelia actually overlapped. Unlike the situation in P. tetraurelia, the timing of IDS in T. thermophila is determined within the same cell cycle, as suggested by a study on a cell shape mutant (conical) in T. thermophila (Doerder et al., 1975; Schafer and Cleffmann, 1982). The point of commitment to IDS occurs early in the T. thermophila cell cycle, about 15 min prior to IDS (Wolfe, 1976). A similar transition point corresponding to PCD was also identified in T. thermophila by heat shock arrest experiments, termed the 'physiological transition point' (Rasmussen and Zeuthen, 1962) or the 'stabilization point' (Frankel, 1962). Heat shock treatments, which normally arrest cell cycle progression if they are administered before this point, cause no delay after this point. 3. Why study cell cycle in ciliates. The features that make ciliates attractive models for investigations of cell cycle regulation can be summarized as follows. First, despite a substantial understanding of the 33 molecular mechanisms of cell cycle regulation in yeast, vertebrates and plants, little is known about ciliates. Given the early evolutionary divergence of ciliates and higher eukaryotes, it is of interest to examine conservation of higher eukaryotic cell cycle regulatory machinery in ciliates. Second, each ciliate cell has a very unique cell structure when compared with most other eukaryotic cells in that two structurally and functionally distinct nuclei reside in the same cytoplasm. As cell division requires precise coordination of distinct cell cycle events, this unique cell structure raises a very important and unique question as to how to coordinate cell cycle events such as D N A synthesis, nuclear divisions and morphogenesis within the ciliate cell. Third, because of the relatively imprecise nature of the amitotic macronuclear division process, and the unusual mechanisms used by either P. tetraurelia or T. thermophila to maintain macronuclear D N A content, initiation of cell division in both ciliates can not be dependent on the precise doubling of macronuclear D N A content. This differs from the situation in typical eukaryotic cells in which checkpoint controls act throughout the cell cycle to ensure that cells do not enter mitosis until D N A replication is complete and cells will not start another round of D N A replication before they have undergone mitosis. Finally, a highly efficient method for synchronizing mass culture of vegetatively growing P. tetraurelia cells has been developed in which cells are selected based on their sizes by centrifugal elutriation (Adl and Berger 1995, Tang et al., 1997). This approach can be adapted for T. thermophila with minimum modifications. The extent of synchrony achieved by this approach is comparable to that of hand-selected dividing cells. No interference with cell cycle events has been observed. 34 Therefore, ciliates offer unique experimental systems to provide further insights into the transition point controls and coordination between various cell cycle events. F. Rationale and research objectives. Since it is well known that Cdk/cyclin complexes are the central regulators of cell cycle events, identification and characterization of ciliate Cdks and cyclins are the first steps in an understanding of the molecular mechanisms underlying these processes. Moreover, it will be interesting to understand what alterations occur to the basic cell cycle elements resulting in the lack of interdependency between macronuclear S phase and cytokinesis during normal ciliate cell cycle. To uncover the molecular machinery underlying ciliate cell cycle regulation, a search for Cdk/cyclin homologues in P. tetraurelia was initiated a few years ago in our laboratory. Western blot analysis with anti-PSTAIRE antibody raised against one of the most conserved region in Cdks suggested the presence of two classes of Cdk-related proteins (Tang et al., 1994). A P. tetraurelia Cdk gene homologue, PtCDKl, was cloned by utilizing a homology-based approach. The gene product corresponded to the larger of the two peptides recognized by anti-PSTAIRE antibody (Tang et al., 1995). Unlike most Cdks, PtCdkl does not bind to pl3™ c /. Its histone HI kinase activity is temporally associated with IDS and macronuclear D N A replication, and the protein is localized exclusively in the macronucleus (Tang et al., 1997). In contrast, the smaller Cdk detected by the PSTAIRE antibody, PtCdk3, binds pl3™ c 7, its histone HI kinase activity peaks at PCD, when the cell becomes committed to cell division. The gene encoding this protein has not been cloned. The regulation of PtCdkl kinase activity during the vegetative cell cycle does not involve oscillations in its transcript or protein level. Major regulatory 35 phosphorylation sites equivalent to Thrl4, Tyrl5 and Thrl61 in human C D K 1 , which are shared among most Cdks, are conserved in the PtCdkl sequence. Regions that are involved in cyclin binding including the PSTAIRE motif in protein kinase domain III (Pines and Hunter, 1990) are also present. It seems possible that the regulation of the kinase activity of the P. tetraurelia Cdk homologue(s) might include binding of cyclins and phosphorylation/dephosphorylation on conserved residues. The objectives of this work were to complete the analysis of the Paramecium cell cycle by: (1), identifying additional Cdk-related protein(s) in P. tetraurelia; (2), identifying the P. tetraurelia cyclin homologue(s); (3), characterizing roles of Cdks and cyclins in the P. tetraurelia cell cycle control. An additional objective was to extend the studies on the cell cycle in Paramecium to other groups of ciliates. Since only one stop codon is used by ciliates, and well-characterized cell cycle mutants are lacking, the high degree of conservation of cell cycle control genes between species suggested a homology-based approach as the route of choice for cloning Cdk and cyclin genes in the ciliates. Given that the best sequence conservation among cyclins was observed in the 'cyclin box' region, PCR primers corresponding to that region were designed for cloning cyclins. To search for additional Cdks in P. tetraurelia, I decreased the PCR annealing temperature to amplify any PtCDKl-related sequences in the P. tetraurelia genome. Following success in cloning both cyclins and the PtCdk2 gene, specific antibodies against both cyclins and PtCdk2 were generated. These antibodies were used to biochemically characterize and analyze their roles in P. tetraurelia cell cycle regulation. I subsequently turned my attention tb a CDK gene in T. thermophila, in which availability of highly sophisticated homologous recombination gene knockout 36 technique makes functional characterization of cell cycle genes in ciliates feasible. Phenotypic analysis of a TtCDKl knockout clone revealed physiological functions of TtCdkl in T. thermophila. 37 M A T E R I A L S A N D M E T H O D S A . Materials. Al l restriction endonucleases and modifying enzymes were purchased either from GIBCO-BRL (Gaithersburg, MD) or from New England Biolabs (Beverly, M A ) . Plasmid Preparation Kits, Lambda D N A Preparation Kit, D N A Gel Purification Kit were products of Qiagen (Santa Clarita, CA). DIG-Labeling and Detection reagents were from Boehringer Mannheim (Mannheim, Germany). Enhanced Chemiluminescence Kit (ECL), radioactive isotopes and Hybond membranes were purchased from Amersham (Arlington, IL). Yeast pl3™ e 7 conjugated to CNBr-activated Sepharose CL4B beads was the product of the Upstate Biotechnology (Lake placid, NY). Protein A agarose beads were from GIBCO-BRL. Glutathione Sepharose 4B was from Pharmacia (Uppsala, Sweden). Immobilon-P membrane was from Millipore (Bedford, MA) . Al l the oligonucleotide primers used for gene cloning and sequencing, and the peptides used for generating antibodies were synthesized by the Nucleic Acid and Peptide Sequencing (NAPS) unit of the University of British Columbia. New Zealand White rabbits used for antibody preparation were purchased through and maintained in the U B C Animal Care Centre. Unless otherwise specified, all other chemicals were purchased from Sigma (St. Louis, MO). B . Cell stocks and culture. Paramecium tetraurelia wild type stock 51-S cells were grown at 27°C in phosphate-buffered wheat grass infusion (Sonneborn, 1970) supplemented with 38 stigmasterol (5 jxg/ml), and inoculated with Klebsiella pneumoniae (ATCC-27889) as the food organism one day before use (Sonneborn, 1970). Tetrahymena thermophila strains CU428.2 [mpr-l/mpr-1 (MPR1, mp-s, VII)] and B2806.2 (II) were grown in Neff growth medium (0.25% proteose peptone, 0.25% Yeast extract 0.55% glucose and 33 p,M FeCh) with shaking at 30°C. Sterkiella histriomuscorum were grown in minimal algal medium supplemented with bacterized infusion (30% v/v) and an inoculum of Chlamydomonas. Belpharisma intermedium and Colpoda sp. were grown in bacterized wheat grass infusion medium. C. Centrifugal Elutriation. Centrifugal elutriation was used to prepare synchronous samples. It was carried out as described by Tang et al., (1994). 2-3xl0 6 Paramecium cells were grown to stationary phase and maintained at 27°C for 5-7 days until more than 90% of the population had completed autogamy. About 36 h before elutriation, the cells were diluted to a density of 600-800 cells/ml with fresh medium and refed every 12 h by the addition of one volume of the freshly inoculated medium. The cell densities were monitored to ensure that the number cells per ml did not exceed 2000, otherwise cells began to starve and cell cycle duration was changed. For elutriation, 1-I.5xl0 7 cells were loaded onto a Beckman elutriator chamber (30 ml) on a JE.5 rotor (Beckman, Palo Alto, CA) at 550 g and at a pump rate of 75 ml/min. Following a 100 ml wash with fresh medium, the fractions collected by stepwise increases of the pump speed were pooled until the composite contained around 5% of the starting cell population. This synchronous population was reinoculated into the fresh medium and maintained at 27°C and harvested at one-hour intervals starting 30 min post elutriation. Cells were harvested by centrifugation at 2,500 39 rpm for 1 min, washed twice with cold Dryl's buffer (Sonneborn, 1970), and were immediately frozen at -70°C for R N A or protein extraction. For T. thermophila, 3x l0 8 log-phase cells were loaded onto the chamber at 650 g and at a pump rate of 35 ml/min. Following a 50 ml wash with Neff growth medium, the fractions collected by stepwise increase of the pump speed were pooled until the composite contained around 5% of the initial cell population. This synchronous population was then kept at 30°C with shaking. Samples were harvested at 30-min intervals starting 30-min post elutriation. After washing twice with phosphate-buffered saline (PBS), cells were processed for protein lysate preparation. D. Genomic D N A Preparation. For genomic D N A isolation, cells were harvested by centrifugation at 2,500 rpm for 1 min from exponentially growing culture, washed twice with Dryl's buffer/PBS and lyzed at 65°C in 100 mM Tris, 50 mM E D T A (pH8.0), 200 mM NaCl, 1% SDS and 1 mg/ml proteinase K (Gibco-BRL) for 3-6 h till lysate became clear. The lysate was extracted three times with phenol: chloroform: isoamyl alcohol (25:24:1), and twice with chloroform only. D N A was then precipitated by ethanol at -20°C for at least one hour. The D N A pellet was washed three times with 70% ethanol, dried in the air and redissolved in TE buffer or sterile distilled water. E . R N A Preparation. Total R N A isolation was performed using the TRIzol Reagent from GIBCO-BRL. Briefly, exponentially growing cells were pelleted by centrifugation and lysed in the Reagent (1 ml of the Reagent per 5x l0 4 of P. tetraurelia cells or 1.2xl05 of T. thermophila cells) by repetitive pipetting. The lysate was incubated at 25°C for 5 min to 40 permit the complete dissociation of nucleoprotein complexes. The sample was then purified with chloroform extraction, and centrifuged at 12,000 g for 15 min at 4°C. Following centrifugation, RNA was precipitated from the supernatant by mixing with isopropyl alcohol. F. Design of Oligonucleotide Primers and P C R Amplification. The following degenerate oligonucleotides were used to amplify the P. tetraurelia mitotic cyclins by PCR: Sense primer: 5'-ATGA/CGAGCA/TATT/AT/CTA/GG/ATA/TGA-3 ' ; Antisense primer: 5'-ATT/CTCT/CTCA/GTAT/C TTT/AG/CA/TT/AGC-3 ' . These primers correspond to two highly conserved regions within the mitotic 'cyclin box', M R A I L V and A S K Y E E I , respectively. P. tetraurelia genomic D N A (1 u,g) was used in a 50 pj PCR reaction consisting of 50 mM Tris-HCI (pH 8.0), 2.0 mM MgCl 2 , 0.05% Tween 20, 0.05% NP-40, 200 u M each of dATP, dTTP, dCTP, dGTP, 100 pmol of each primer, and 2.5 U Taq D N A polymerase (GIBCO-BRL). After an initial 5-min denaturation at 94°C, 30 cycles of amplification were carried out (1 min at 94°C, 2 min at 50°C and 3 min at 72°C). PCR products of the predicted length (-200 bp) were isolated by agarose electrophoresis, purified with Qiagen Gel Purification Kit, and subcloned into the plasmid pBluescript II KS+ (Stratagene, Cambridge, UK). The resulting plasmids were subjected to D N A sequencing by the dideoxy-mediated chain-termination method (Sambrook et al., 1989). For PtCDK2 and TtCDKl, degenerate oligonucleotide primers were designed based on the two conserved regions of known Cdk sequences. The sense primer, based on G E G T Y G (domain I of protein kinases), was 5'-GGA/TGAA/GGGA/TACA/TTAT/CGG-3 ' . The antisense primer, based on D L K P Q N 41 (domain VI), was 5 ' -G/ATTT/CTA/GA/TGGC/TTTA/TAA/GG/ATC-3 ' . For PtCDK2, PCR was carried out at an annealing temperature of 37°C instead of 50°C with P. tetraurelia genomic D N A as template. A product of -400 bp was amplified, which was then purified with Qiagen Gel Purification Kit and subcloned into the plasmid pBluescript II KS+. The resulting plasmids were subjected to D N A sequencing by the dideoxynucleotide chain termination method. For TtCDKl, first strand cDNA synthesized from total R N A by an oligo-dTn primer (see Appendix I) was used as template. PCR was run at an annealing temperature of 50°C. G . D N A library screen. Two P. tetraurelia D N A libraries were employed to obtain the full-length sequence of PtCYCla: a A,gtl 1 cDNA library constructed using poly(A)+ R N A from vegetative P. tetraurelia cells, kindly provided by Dr. James Forney (Purdue University); a genomic library constructed in XEMBL3 with Sau3A partially digested P. tetraurelia genomic D N A which was a gift from Dr. Eric Meyer (Laboratoire de Genetique Moleculaire, Ecole Normale Superieure, Paris). Approximately 1,000,000 plaques from each library were screened with a 32P-labeled D N A probe corresponding to the initial PCR fragment, as described by Sambrook et al. (1989). H . 3' and 5' R A C E s . 3' and 5' RACEs (Rapid Amplification of cDNA ends) (Frohman, 1990) were used to obtain PtCDKl, PtCYClb, PtCYC2 and TtCDKl cDNA full-length sequences. Briefly, for the 3 ' end sequence, first strand cDNA was synthesized from total R N A by an oligo-dTn primer and then was used as template in the PCR reaction primed by oligo-dTn and a gene specific sense primer derived from the original PCR fragment. For the 5' end 42 sequence, first strand cDNA was made from total RNA by a gene-specific antisense primer, and then tailed with dAs with Terminal d Transferase (Gibco-BRL). The resulting cDNA was amplified with oligo-dTn primer and an internal gene-specific antisense primer. Detailed sequence information on these primers can be found in Appendix I. The complete cDNA and genomic D N A sequences were finally isolated by PCR using primer pairs derived from both ends of the sequence. D N A sequencing was carried out on both strands. I. P r e p a r a t i o n o f D N A P r o b e . The 32P-labeled D N A probe used in the library screen was prepared using a Random Hexamer Priming kit from Boehringer Mannheim (Mannheim, Germany) in the presence of [a- 3 2P]dATP (Amersham). DIG (Digoxigenin)-labeled D N A probes used in genomic Southern blot analysis were prepared by PCR by including DIG-11-dUTP (Boehringer Mannheim) in the reaction mix, as described by An et al. (1992). J. G e n o m i c S o u t h e r n B l o t A n a l y s i s . Thirty micrograms of genomic D N A digested with restriction enzymes were subjected to electrophoresis on 0.7% agarose gel and blotted onto Hybond-N+ membrane (Amersham) using downward transferring technique in 0.4 M NaOH (Koetsier et al., 1993). The membrane was hybridized to DIG-labeled D N A probes, detected and stripped as described by Engler-Blum et dl. (1993). The membrane was washed at 40°C and 65°C to provide low and high stringency conditions, respectively. K . P r o t e i n L y s a t e P r e p a r a t i o n . Cells were lysed in 4 volumes of lysis buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 50 mM NaF, 5 mM EDTA, 1 mM DTT, 0.1% NP-40), supplemented with a cocktail of protease inhibitors including 50 p.g/ml PMSF (phenylmethyl sulfonyl floride), 2 43 u.g/ml leupeptin, 4 ng/ml aprotinin, and 1 p-g/ml pepstatin A. The lysate was incubated on ice for 15 min, and centrifuged at 18,400 g for 15 min at 4°C. The supernatant was kept at -20°C. An aliquot was taken for protein quantitation using the Bradford methods (Bradford, 1976). L. Generation of Polyclonal Antibodies against Cdks and Cyclins /. Preparation of antiserum against P. tetraurelia Cdk2. Polyclonal antiserum was raised against synthetic peptide corresponding to residues 240-255 of PtCdk2 with additional cysteine at its amino terminus (CDFKSTFPRWPTPTNPA) to facilitate coupling of the peptide to keyhole limpet hemocyanin (KLH). Two hundred fifty micrograms of peptide in RIBI adjuvant (Sigma) was injected intramuscularly into New Zealand White rabbits in the first injection followed by boosts with 200 p:g of peptide every 4 weeks. 2. Preparation of anti-PtCycl and anti-PtCyc2 antisera. A BamHl-EcoRl PtCYCla cDNA fragment, containing residues 1-68 of the coding region, was subcloned in-frame into a pGEX-2T vector (Pharmacia, Uppsala, Sweden), and the plasmids were transformed into bacteria. GST-PtCycl protein synthesis was induced by 0.2 mM IPTG, and the protein was purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B (Pharmacia) (Smith and Johnson, 1988). GST-PtCycl fusion protein was emulsified with RIBI adjuvant, and injected intramuscularly into rabbits. Approximately 0.7 mg of protein was used per injection. A peptide derived from the carboxyl end of PtCyc2, corresponding to residues 321-336 with an additional cysteine at its amino end (CQEVSRIRVERQIKQQK) was 44 synthesized and coupled to K L H (keyhole limpet hemocyanin), and then used for injection into rabbits for generating anti-PtCyc2 antibody. 3. Preparation of antiserum specific for T. thermophila Cdkl. A peptide derived from the carboxyl terminus of T. thermophila Cdk l , corresponding to residues 303-318 with an additional cysteine at its amino end (CSREDIAKFEPNQVHMY) was synthesized and coupled to keyhole limpet hemocyanin (KLH). Two hundred micrograms of peptide in Freund's adjuvant (Sigma) were injected into rabbits every 4 weeks until anti-PtCdkl immunoreactivity could be detected by Western blot analysis. The antibody production was carried out as described by Harlow and Lane (1988). M . Western Blot Analysis. Western blotting was carried out as described by Harlow & Lane (1988). Briefly, proteins were resolved on 12.5% SDS-PAGE and transferred to Immobilon-P membrane (Millipore). After transfer, membrane was first incubated with blocking buffer (5% non-fat milk and 0.1% Tween-20 in PBS) for 1 h, and then with primary antibody (1:500 dilution) for 1 h. After three washes in blocking buffer, the primary antibody was revealed by enhanced chemiluminescence (ECL, Amersham) using horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1:4000 dilution). The intensity of bands on X-ray films was quantitated using the public domain NIH image program (developed by The U.S. National Institutes of Health, Bethesda, MD). N . Immunoprecipitation. Protein lysate (500 u,g) was first pre-cleared with 50 u,l of protein A-agarose beads (Gibco-BRL) (50% v/v in lysis buffer) for 2 h at 4°C. The supernatant was then incubated 45 for 4 h with antiserum (5 ul per ml of lysate) or the corresponding pre-irnmune serum, followed by 1 h of incubation with 40 u.1 of protein A-agarose (50% v/v) with constant rotation at 4°C. Beads were pelleted by centrifugation and washed three times with lysis buffer. For immunoblotting, the pellets were resuspended in 20 ul of 2x Laemmli sample buffer (Laemmli, 1970) and heated at 100°C for 3 min. O. pl3 s u e 7 Binding Assay. Yeast pl3™ c ; conjugated to CNBr-activated Sepharose CL4B was purchased from the Upstate Biotechnology (Lake placid, NY) . The concentration of coupled pl3™ c ; was 3.7 mg/ml. Just before use, 80 ul of pl3™ c / beads (50% v/v) was washed with 1 ml of bead buffer (50 mM Tris pH7.4, 250 mM NaCl, 5 mM NaF, 5 mM EDTA, 5 mM EGTA, 100 mM Benzamidine, 0.1% NP-40) and resuspended in 400 ul bead buffer. The protein lysate (250 p:g in a volume of 400 ul) was added to the beads and kept under constant rotation at 4°C for 1 h. After a brief centrifugation, the beads were washed five times with 1 ml of bead buffer. The beads were then resuspended in 40 ul of 2x Laemmli sample buffer and boiled for 3 min before loading onto the SDS-PAGE. In the pl3™ c / immunodepletion experiment, multiple rounds of pl3™ c ; binding were performed as described above in order to deplete all pl3'™e/ bound Cdk(s), except that 40 ul of packed beads was used instead of 80 ul of 50% (v/v) bead suspension. After each round of the binding, beads were pelleted, washed, and the bound proteins were eluted by boding in 2x Laemmli sample buffer. In the meantime, an aliquot of 15 ul of supernatant after each round of the binding was saved, and then a fresh batch of pl3™ c ; beads (40 ul) was added to the remaining supernatant. Three rounds of p l3 s " c ; binding 46 experiments were carried out. Equal volume (15 pi) from all three bound fractions (precipitation, PI , P2, P3) and three unbound fractions (supernatant, SI, S2, S3) were resolved on SDS-PAGE and then immunoblotted with either anti-PSTAIRE, anti-PtCdkl or anti-PtCdk2 antibodies. P. Histone HI kinase assay. Six hundred micrograms of total protein lysate was first pre-cleared with 50 pi of protein A-agarose beads (GIBCO-BRL) (50% v/v in lysis buffer) for 30 min at 4°C. The supernatant was then incubated with 2 of of antiserum or corresponding pre-immune serum on ice for 3 h, followed by 1 h of incubation with 50 pi protein A-agarose beads (50% v/v) with constant rotation at 4°C. Beads were washed five times in lysis buffer and twice with l x kinase assay mix (50 mM Tris, pH7.5, 10 mM MgCl 2 , 1 mM DTT, 40 u M ATP, 1 mM EGTA). Then, 10 pi of kinase assay cocktail containing 0.2 mg/ml bovine histone HI (StressGen, Victoria, CA) and 320 pmol (10 uCi) [y-32P] ATP (Amersham) was added and the reactions were incubated at 27°C for 20 min. The reaction was terminated by addition of equal volume of 2x Laemmli sample buffer. Aliquots were separated on SDS-PAGE. Phosphorylated histone HI was detected by autoradiography and quantitated by liquid scintillation counting of excised histone HI bands. Q. Expression of G S T -P tCdk2 fusion proteins in bacteria. A BamHl-EcoRI cDNA fragment containing residues 212 to 301 of PtCdk2 was subcloned into a pGEX-2T vector (Pharmacia) and transformed into DH5oc bacteria. The resulting clones were confirmed by D N A sequencing on both strands. Production of recombinant GST-Cdk2 fusion protein was induced by 0.2 mM IPTG. Proteins were 47 purified by affinity chromotography using Glutathione Sepharose 4B as described by Smith and Johnson (1988). R. Indirect Immunofluorescence Microscopy. Immunofluorescence staining was performed as described by Williams and Nelsen (1997). For single staining, T. thermophila cells were permeabilized and fixed in 50% ethanol containing 0.2% Triton X-100 for 30 min on ice. The samples were rinsed twice in Tris-buffered saline (TBS, pH8.2) containing 0.1% bovine serum albumin (BSA), and then incubated in anti-TtCdkl antiserum at a dilution of 1:100 in TBS containing 1% B S A for 1 h at room temperature. After three washes in 0.1% BSA-TBS, the samples were suspended in FITC-conjugated goat anti-rabbit IgG (Amersham) at a dilution of 1:100 for 1 hr. The cells were treated with RNase (10 pg/ml) before being counterstained with propidium iodide (1 pg/ml). The samples were mounted in 90% glycerol in PBS (pH8.0) containing 2.5% D A B C O (Sigma). For double staining, anti-TtCdkl antiserum (1:100) and anti-r. thermophila a-tubulin monoclonal antibody 15D3 (1:5) (kindly provided by Dr. Joseph Frankel, University of Iowa, Iowa City, IO) were used as primary antibodies. FITC-conjugated goat anti rabbit IgG and Texas Red-conjugated donkey anti mouse IgG (1:100 each) (Amersham) were used as secondary anitbodies. Immunolabelled cells were observed under a Bio-Rad M R C 600 or Radiance Plus confocal laser fluorescence microscope equipped with a Kr/Ar laser. S. Targeted Disruption of Macronuclear TtCDKl by Biolistic Transformation. A pBluescript-based plasmid, pTtCDKl, containing the 1.7-kb TtCDKl genomic D N A sequence, was linearized at the single Hindi site within its coding region to 48 construct a targeting plasmid for disruption of the TtCDKl gene. The neomycin-resistance gene cassette that contains the histone H4 promoter region, the neomycin-resistance coding region, and the transcription terminator of P-tubulin gene was inserted into this site using the T)4T2-lAHindIII plasmid (a gift from Dr. Martin Gorovsky, University of Rochester, Rochester, N Y ; Gaertig et al., 1994) to generate the knockout plasmid, pTtCDKl-neo. Single strand D N A sequencing was performed to select the clones in which the neomycin-resistance gene cassette was inserted in the transcriptional orientation of the TtCDKl gene. The disruption plasmid was digested to release the Notl-Hindlll fragment containing neomycin-resistance cassette flanked by the TtCDKl gene sequences. The mixture of digestion reaction was used to coat tungsten particles (Sanford et al., 1993). For Holistic transformation of the macronucleus (Cassidy-Hanley et al., 1997), B2806.2 and CU428.2 were starved in 10 mM Tris-HCl (pH 7.5) for 14 h on a 30°C shaking incubator prior to mating. Ten hours after the initiation of conjugation, which is the time of development of new macronuclear anlagen in progeny cells, paired cells were bombarded with disruption plasmid or control plasmid (pBluescript only) coated tungsten particles (0.7 um) at 900 psi, using DuPont Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, Richmond, CA). After bombardment, the cells were recovered in Neff medium for 7 h at 30°C, then plated into Neff supplemented with paromomycin at a concentration of 20 ug/ml. Approximately 6-7 days after bombardment, paromomycin resistant clones were picked up and replated into Neff medium containing increasing concentrations of paromomycin (ie. 50, 100, 150, 500 and 1000 ug/ml). T. Flow Cytometry Analysis. 49 Logarithmatically growing Tetrahymena cells were lysed in 0.25 M sucrose, 10 mM MgCl 2 , and 0.5% NP-40 at a concentration of 1.5xl06 cells/ml. After cell lysis, propidium iodide was then added to 50 pg/ml to stain nuclei for 1 h before flow cytometry analysis using a Becton Dickinson flow cytometer (BD Biosciences, San Jose, CA) (Wei et al., 1999). Flow cytometry analysis was performed at the Biomedical Research Centre, UBC. 50 C H A P T E R 1. A N O V E L M E M B E R OF T H E CYCLIN-DEPENDENT KINASE F A M I L Y IN PARAMECIUM TETRA URELIA 1.1 INTRODUCTION In budding and fission yeast, both the Gl -S and the G2-M transitions are controlled by the same p34 c d c 2 / C D C 2 S kinase. Various functions of the p34 c t f c 2 / C D C 2 S kinase are performed by varieties of the kinase that differ mainly, if not solely, in their cyclin subunit. In mammals, a variation of this theme is used for cell cycle control. As in yeast, multiple cyclins (thus far 16) have been identified (Johnson and Walker, 1999; Table 1). In contrast to yeast, the CDK1 is not the only catalytic subunit that can interact with cyclins. While CDK1 is essential for the G2-M transition in vertebrate cells, at least 8 more related sequences have been reported, even though functions for some of them remain to be established. It is clear that each member of this multigene family has separate and distinct functions in the control of cell growth, division, differentiation and/or development. Coordination of the activities of these Cdks may be essential to control the varied and distinct cell cycle events and signaling pathways found within metazoan organisms. In previous studies, Western blot analysis with anti-PSTAIRE antibodies revealed the presence of two size classes of Cdk-related proteins in Paramecium tetraurelia and Tetrahymena thermophila (Fujishima et al., 1992; Tang et al, 1994). In P. tetraurelia, the major Cdk, PtCdkl, whose encoding gene has been sequenced, does not bind to pl3™ c /, whereas the minor one binds to pl3™ c /. The PtCdkl associated histone HI kinase activity seems to be involved in the control of macronuclear D N A synthesis (Tang et al, 1997). The pl3™ c i associated histone HI kinase activity peak was observed at PCD, when the cell becomes irreversibly committed to division (Tang et al, 1994). This suggests that 51 the two kinases are responsible for the control of different cell cycle events in P. tetraurelia. In an attempt to identify other CD^T-related genes in P. tetraurelia, I reduced the stringency of the PCR reaction and cloned PtCDK2. Surprisingly, PtCdk2 does not bind to pl3™e 7 and exhibits histone HI kinase activity at the end of cell cycle, suggesting it is a novel class of Cdk in P. tetraurelia. 1.2 R E S U L T S 1.2.1 Primary sequence predicts that PtCdk2 is a cyclin-dependent protein kinase. Two degenerate oligonucleotide primers representing two conserved regions were designed by comparison of the amino acid sequences of known Cdks, and used in PCR with P. tetraurelia genomic D N A as template. The PCR was carried out at low annealing temperature of 37°C in order to pick up PtCDKl-related genes. On agarose gel, the major PCR product was observed as a -400 bp band, which was in the size range expected for products derived from Cdk homologues with this primer pair. After subcloning into pBluescript KS+ and blue/white selection, 15 positive colonies were obtained. Twelve of them contained the sequences corresponding to one or the other of the two gene isoforms of CDK1 identified in previous work (Tang et al., 1995). Three others were novel and identical. Their putative amino acid sequences contained EGVPSTAIREISLLKE ('PSTAIRE') motif characteristic of the Cdk family, suggesting they might encode a novel Cdk in P. tetraurelia. I tentatively call this new Cdk homologue PtCDKl. The full-length cDNA sequence for PtCDKl was obtained by 3' and 5' R A C E anchor PCRs (Frohman, 1990), as described in Materials and Methods. Even though no in-frame stop codon (TGA) was found upstream of the presumptive initiation codon A T G , 52 the sequence is likely to represent the complete open reading frame (ORF) of PtCDK2, considering that its size is close to that of PtCDKl. Moreover, a single 1.2 kb transcript was observed for PtCDKl (data not shown), which is similar to the size of PtCDKl transcript (1.3 kb) (Tang et al, 1995). The putative ORF is predicted to encode a protein of 301 amino acids with all protein kinase catalytic domains, suggesting that PtCdk2 is a protein kinase (Fig. 1-1). The 'EGVPSTAIREISLLKE' ( 'PSTAIRE' region) in PtCdk2 matches perfectly with the canonical sequence in ^ 2>Acdc2/CDC28 of yeast whereas one amino acid substitution was observed in PtCdkl (valine to isoleucine) (Tang et al, 1995). No putative poly-adenylation signal near the 3' end was identified, as is the case for most of the Paramecium genes cloned so far (Prescott, 1994). PtCdk2 is 7 amino acid residues shorter than PtCdkl. The predicted relative molecular mass of PtCdk2 is 35 kDa, which matches that of the minor polypeptide recognized by anti-PSTAIRE antibody in a previous study (Tang etal, 1994). The Paramecium genetic code is different from the universal genetic code used by most organisms (Caron and Meyer, 1985; Horowitz and Gorovsky, 1985; Preer et al, 1985), in that it uses U A A and U A G as glutamine codons instead of as stop codons. Within PtCDKl ORF, there are 10 U A A coded glutamines and 2 U A G coded glutamines, suggesting a strong preference for U A A (Fig. 1-1). This is consistent with the previous findings by Martindale (1989). 53 ATTAAAATTAACGAATAGCTGTTAGTT -1 ATGGATTTAGCATAAAGCGAAGAAAGATATTAAAAATTGGAAAAAATAGGAGAAGGAACATATGGATTA 6 9 M D L A Q * S E E R Y Q * K L E K I G E G T Y G L 23 GTATACAAAGCAAGAGATAATCAAACTGGCGATATAGTGGCATTAAAAAAAATAAGAATGGATCATGAA 13 8 V Y K A R D N Q T G D I V A L K K I R M D H E 46 GACGAAGGTGTTCCATCTACTGCAATTAGAGAAATTTCATTATTAAAAGAAgtaaaattgtattaatca 207 D E G V P S T A I R E I S L L K E 63 tgaaagGTTCAACATCCAAATATTGTTCCACTGAAAGATGTAGTTTATGATGAATCAAGATTATATTTA 27 6 V Q H P N I V P L K D V V Y D E S R L Y L 84 ATATTTGATTTTGTAGATTTAGATCTAAAAAAATATATGGAGAGCGTACCTTAGTTAGATAGAATGCAA 3 45 I F D F V D L D L K K Y M E S V P Q * L D R M Q 107 GTGAAGAAGTTCATAAATTAAATGATACAAGCCTTAAACTATTGTCATTAAAATAGAGTCATTCACAGA 414 V K K F I N Q * M I Q A L N Y C H Q * N R V I H R 130 GACTTAAAACCATAAAATATCCTAGTAGATATCAAATAATAAAATACCTAAATCGCAGATTTCGGATTG 483 D L K P Q* N I L V D I K Q * Q * N T Q * I A D F G L 153 GCTAGAGC ATTTGGCTTACCTTTAAAAACTTACACCCATGAAGTTATAACCCTATGGTATAGAGCCCCT 552 A R A F G L P L K T Y T H E V I T L W Y R A P 176 GAGATTTTACTTGGGTAAAGATAATACTCAACACCAGTAGATATTTGGTCATTGGGATGCATCTTTGCT 621 E I L L G Q * R Q * Y S T P V D I W S L G C I F A 199 GAAATGGCACAAAAAAGACCATTATTTTGTGGAGATTCAGAAATAGATTAGTTATTTAAGATATTTAAA 690 E M A Q K R P L F C G D [ S E I D Q * L F K I F K 222 ATTATGGGCACTCCTAAAGAATCTACTTGGCCTGGTGTTAGCACTTTACCTGACTTTAAGAGTACCTTC 759 I M G T P K E S T . W P G V S T L P D F K S T F 245 CCAAGATGGCCAACCCCTACTAATCCAGCTGCAACGTTAGGAAAAGATATTACTAATTTATGTCCACTA 828 P R W P T P T N P A A T L G K D I T N L C P L 268 GGATTAGATCTCCTTTCTAAAATGATAACATATGATCCTTATGCTAGAATCACTGCAGAGGAAGCCCTA 897 G L D L L S K M I T Y D P Y A R I T A E E A L 291 AAACATGCTTATTTTGATGAATTAAATAACTGATATTAAACCCAGAATACTAAATTATTTGATAAAAAA 966 K H A Y . F D E L N N * ] 301 AAAAAAAAA 975 Fig. 1-1. Nucleotide sequence of PtCDKl, and the predicted amino acid sequence of the gene product, PtCdk2. The conserved 'PSTAIRE' region is double underlined. Putative phosphorylation sites, Thr20, Tyr21 and Thrl65 in PtCdk2, equivalent to Thrl4, Tyrl5 and Thrl61 of human CDK1, respectively, are indicated by bold italics. A 24-nucleotide intron in the sequence is in lower case. Q* is glutamine encoded by T A A or T A G . "*" indicates the stop codon TGA. The sequence used for designing the antigenic peptide is single underlined. The region that was expressed as GST fusion protein is in parentheses '[ ]'. The GenBank accession number of this sequence is AF126147. 54 Comparison of PtCDK2 genomic and cDNA sequences revealed a single intron of 24 bp located just downstream of the 'PSTAIRE' region (Fig. 1-1). Its position does not correspond to that of either of the two introns in PtCDKl (Tang et al., 1995). Small introns are very common in the, Paramecium genome (Russell et al., 1994). The PtCDK2 intron has consensus 573' splice sites GT/AG without apparent branch point consensus sequences. Phosphorylation and dephosphorylation of specific residues regulate the kinase activity of Cdks (Krek and Nigg, 1991, 1992; Norbury et al., 1991). Homologues to the regulatory phosphorylation residues, Thrl4, Tyrl5 and Thrl61 in human CDK1, Thr20, Tyr21 and Thrl65, are conserved in PtCdk2 (Fig. 1-1). Therefore, PtCdk2 has the potential to be regulated in an analogous fashion to human C D K L Southern blot analysis of Paramecium genomic D N A digested by EcoRI, Xbal, Pstl and Hindlll, with the DIG-labeled D N A probe derived from the full-length PtCDK2 coding region showed two hybridizing bands in each lane under high stringency conditions, suggesting that there are at least two copies of PtCDK2 gene in the Paramecium genome (Fig. 1-2A). Similarly, there are at least two bands when the same blot was stripped and reprobed with the PtCDKl D N A probe (Fig. 1-2B). No recognition sites for the above restriction enzymes were found in both sequences. However, the possibility of those less intense bands in each lane corresponding to the micronuclear copy of the genes can not be eliminated. Furthermore, genomic Southern blot analysis results suggest that PtCDKl and PtCDKl do not reside tandemly in the genome, as then-hybridizing patterns differ greatly. 55 1 2 3 4 kb 11.0 — 9.0 — 8.0 7.0 6.0 ~ ~ 5.0 4 0 — 3.0 — 1.0 0.5 •••^  2.0 — ^ 1.6 — A B Fig. 1-2. Genomic Southern blot analysis of Paramecium CDK genes. Paramecium genomic D N A was digested with EcoRI (lane 1), Xbal (lane 2), PstI (lane 3), Hindlll (lane 4), and probed with the DIG-labeled PtCDAT2 probe (A). The membrane was then stripped and reprobed with the DIG-PtCDKl probe (B). Arrowheads indicate faint bands on the blot. The 1-kb D N A marker from Gibco-BRL are indicated on the left. 56 1.2.2 PtCdkl exhibits extensive homology to Cdk homologues from other eukaryotes I compared the putative amino acid sequences of PtCdk2 and PtCdkl with Cdks from humans (Lee and Nurse, 1987), Schizosaccharomyces pombe (Hindley and Phear, 1984), Saccharomyces cerevisiae (Lorinze and Reed, 1984), Zea mays (Colasanti and Sundaresan, 1991), Dictystelium discoideum (Michaelis and Weeks, 1992), Plasmodium falciparum (Ross-MacDonald et al., 1994), Entamoebae histolytica (Lohia and Samuelson, 1993) and Trichomonas vaginalis (Riley et al., 1993) (Fig. 1-3). The highest similarity of PtCdk2 was found with Z. mays p34 c d c 2 (61%) and the lowest was found with T. vaginalis (33%). The sequence identity between PtCdkl and Cdks from other eukaryotes ranged from 41% to 60%. Strikingly, the two Paramecium Cdks share only 48% identity with each other, suggesting that the evolutionary separation of PtCdkl and PtCdk2 is ancient and they may have distinct functions in the Paramecium cell cycle regulation. 57 Fig. 1-3. Comparison of the amino acid sequence of Cdk2 and Cdkl from Paramecium tetraurelia with C D K homologues from Trichomonas vaginalis (Tv), Entamoeba histolytica (Eh), Plasmodium falciparum (Pf), Dictyostelium discoideum (Dd), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Zea mays (Zm), and Homo sapiens (Hs). The multiple alignment was generated using the C L U S T A L W program (Higgins et a l , 1994). The boxshade graphic was created with the default parameters of the B O X S H A D E 3.21 program from ISREC (Swiss Institute for Experimental Cancer Research). The black shading indicates 50% or greater amino acid identity, whereas the gray areas 50% or greater amino acid similarity. '*' indicates the residues involved in pl3™ e 7 binding in human C D K l . 58 Cdk2 Cdkl Tv Eh Pf Dd Sc Sp Zltl Hs GIVYK £JVF Gwg GWY GWY Cdk2 Cdkl Tv Eh Pf Dd Sc Sp Zm Hs Cdk2 128 Cdkl 130 Tv Eh Pf Dd Sc Sp Zm Hs 123 122 122 126 133 131 124 125 I H R D L K P Q L H R D L K P Q V H R D I K P E L H R D M K P Q L H R D L K P Q L H R D L K P Q L H R D L K P Q I H R D L K P Q L H R D L K P Q : L H R D L K P Q : Cdk2 198 Cdkl 199 Tv Eh Pf Dd Sc Sp Zm H S P T P T N P Q g Q P D N Cdk2 267 Cdkl 264 Tv Eh Pf Dd Sc Sp Zm Hs AA|^KrArajffc QAKF C g R ^ - Y N K J j j j F g R c | -JSQS—jjgswj - B H K S — B a w G i a JPEQVKKLYVNVK H J P ^ N K P N N N -PSFF nj2j pogs JQQNJ^ RRFH M f e w O 3NQIKKM 59 1.2.3 PtCdk2 corresponds to the polypeptide of 35 kDa recognized by anti-PSTAIRE antibody. To study the temporal regulation and biological activity of PtCdk2, a rabbit antiserum was raised against a peptide corresponding to residues 240-255 of PtCdk2 (Fig. 1-1), a sequence not found in PtCdkl, and was used for all subsequent experiments. Immunoblot analysis of the P. tetraurelia protein lysate showed that the antibody recognized a protein of 35 kDa, corresponding to the minor polypeptide recognized by anti-PSTAIRE antibody (Fig. 1-4, lanes 1 and 3) and matching the predicted molecular mass of PtCdk2. Preincubation of the serum with excess antigenic peptide abolished the immunoreactivity, implying that the antibody was specific for epitopes within the antigenic peptide (data not shown). Moreover, the antibody showed no cross-reaction with PtCdkl (Fig. 1-4, lane 2 and 3). To further confirm the specificity of the antibody, I expressed a partial PtCDK2 sequence (residues 212 to 301) as glutathione-S-transferase (GST) fusion protein in E. coli (Fig. 1-1). Within this region, the only U A A / U A G coded glutamine was Q 2 1 6 , which was later converted to C A A , the conventional glutamine codon by including a mismatch in the sense primer used for subcloning the D N A fragment into pGEX-2T vector. The antigenic peptide used for generating anti-PtCdk2 antibody was also located in this region. After purification on glutathione-Sepharose beads, the fusion protein was resolved on SDS-PAGE as a large band of predicted molecular mass for fusion protein (36 kDa), and a small band of 26 kDa representing GST portion after Coomassie Brilliant Blue staining (data not shown). The presence of GST only protein in the expression products might be due to the different codon usage for P. tetraurelia and E. coli (Martindale, 1989; Sharp et 60 al., 1988). On an immunoblot of the GST-PtCdk2 fusion protein and GST only protein, anti-PtCdk2 peptide antibody recognized only GST-Cdk2 protein (36kDa), and not GST (26 kDa) (Fig. 1-5A), whereas anti-GST antibody recognized both proteins (Fig. 1-5B). These results further support that anti-PtCdk2 peptide antibody is specific for the protein encoded by PtCDKl, 1.2.4 pl3sucl-binding property of PtCdkl reveals a novel class of p34cdc2 homologue in P. tetraurelia. Previous results have shown that PtCdkl does not bind to yeast pl3'™c7 (Tang et al., 1994). However, the minor polypeptide of 35 kDa recognized by anti-PSTAIRE antibody did bind to pl3™ c ; (Fig. 1-6A), and histone HI kinase activity was detected in pl3sucl precipitates, peaking at PCD (Tang et al., 1994). To test whether PtCdk2 corresponds to the p35 polypeptide bound to pl3sucl, a lysate from exponentially growing P. tetraurelia cells was incubated with pl3™ c ;-Sepharose beads. After extensive washing, proteins from both supernatant and beads were resolved on a SDS-PAGE and transferred to Immobilon membrane, and then probed with anti-PtCdk2 antibody. Surprisingly, I found that PtCdk2 remained in the supernatant, as PtCdkl did, suggesting that it is incapable of pl3™ c 7 binding (Fig. 1-6B). As a positive control, p34 e d c 2 from S. pombe was only observed on the pl3™c 7 61 kDa 1 2 3 47.7 — Fig. 1-4. Detection of Cdk homologues in Paramecium lysate. Lysate prepared from asynchronous cells was analyzed by immunoblotting with anti-PSTAIRE antibody (lane 1), anti-GST-PtCdkl fusion protein antibody (lane2) and anti-PtCdk2 peptide antibody (lane 3). Molecular marker from Bio-Rad (Hercules, CA) is indicated on the left. kDa 1 2 1 2 47 .7 -34.6 — -Cdk2 19.2 — A B Fig. 1-5. Specificity of the antibody against PtCdk2 peptide. Lane 1, purified GST-PtCdk2 protein. Lane 2, purified GST without the attached fusion protein. The immunoblot was first probed with anti-PtCdk2 peptide antibody (A). Then, it was stripped and re-probed with anti-GST antibody (B). 62 beads, not in supernatant. (Fig. 1-6C). Based on this observation, it was hypothesized that the p35 polypeptide band recognized by anti-PSTAIRE antibody might consist of two different classes of Cdk homologues with very similar molecular masses, one binding to pl3™ c 7, the other not. The former corresponds to the kinase activity associated with PCD, the latter is PtCdk2. To examine this possibility, a pl3™ c / immunodepletion experiment was carried out. As shown in Figure 1-7,1 observed that only two rounds of incubation with pl3'™c7 were sufficient to deplete all p35 associated with pl3™ e / (Fig. 1-7A: PI , P2, and P3). The third round of incubation brought virtually no 35 kDa polypeptide down, but a p35 polypeptide could be detected by anti-PSTAIRE antibody in the supernatants at all steps (Fig. 1-7A: S l , S2, and S3). These results support the conclusion that p35 band recognized by anti-PSTAIRE antibody, in fact, contains two classes of polypeptides. The p35 polypeptide retained in the supernatants corresponded to PtCdk2 (Fig. 1-7C). As a control, PtCdkl could only be detected in the supernatants (Fig. 1-7B), in agreement with previous results (Tang et ah, 1994). The pl3' v" c / immunodepletion results suggested that PtCdk2 is a new member of the Paramecium Cdk family in addition to PtCdkl and the p l 3 i u c / binding kinase. PtCdk2 has similar molecular mass of 35 kDa to the pl3' s" c ; binding kinase. However, PtCdk2, like PtCdkl, does not bind to pl3™ e /. For convenience, the p35 kinase that binds to pl3™ c ; was named PtCdk3, and the one identified in this study that does not bind pl3™ c 7 PtCdk2, consistent with the newly proposed ciliate genetic nomenclature (Allen et al., 1998). \ 63 W P S W P S W P S • m^ — mm*^. p tCdk2 — p3Vdc2 Anti-PSTAIRE Anti-PtCdk2 Anti-PSTAIRE A B C Fig. 1-6. pl3sucl affinity of the Paramecium Cdks. Panels A and B show protein lysate from P. tetraurelia; Panel C shows protein lysate from S. pombe. W: whole lysate; P: bound fraction eluted from pl3™e 7 beads (pellet); S: unbound fraction (supernatant). Proteins were resolved on SDS-PAGE and immunoblotted with antibodies as indicated. W P1 S1 P2 S2 P3 S3 Anti-PSTAIRE A W P1 S1 P2 S2 P3 S3 Anti-PtCdk1 B W P1 S1 P2 S2 P3 S3 Anti-PtCdk2 «MN» Fig. 1-7. p i 3™'' immunodepletion experiment showing PtCdk2 is a novel Cdk in P. tetraurelia. Aliquots of resulting bound fractions (PI, P2, P3) and unbound fractions (S l , S2, S3) after each round of pl3iucl incubation were immunoblotted with anti-PSTAIRE antibody (A), anti-PtCdkl (B), and anti-PtCdk2 (C). W: whole lysate. 64 1.2.5 PtCdk2 protein level does not vary during the Paramecium vegetative cell cycle. To study PtCdk2 protein levels during the cell cycle, exponentially growing Paramecium cells were synchronized by selecting a population of the smallest, newly divided daughter cells (predominantly in early G l phase) using a Beckman centrifugal elutriation rotor. Initiation of D N A synthesis routinely occurred about 2-2.5 hours after elutriation, as determined by monitoring the incorporation of [3H]-thymidine into macronucleus (Berger, 1971; Tang et al., 1997). The median time of cell division in elutriated samples ranged from 8.5 to 9.5 h post elutriation. The extent of synchrony obtained by centrifugal elutriation was comparable to that of hand-selected dividing cells from the same culture (Adl and Berger, 1995; Tang et al., 1997). Immediately after elutriation, cells were reinoculated into fresh medium and allowed to proceed through one relatively synchronous cell cycle and were sampled at one-hour intervals, starting 30 min after elutriation. To examine the pattern of PtCdk2 protein expression during the vegetative cell cycle, immunoblot analysis with anti-PtCdk2 antibody was performed at each time point when samples were collected. PtCdkl was used as an equal loading control (data not shown). As shown in Figure 1-8A, PtCdk2 appeared as a single band of 35 kDa, and was expressed at roughly comparable levels throughout the vegetative cell cycle, just like PtCdkl (Tang et ai, 1997). I also examined PtCdk2 protein expression in starved cells. Although it was still detectable, PtCdk2 protein was present at much lower level in starved cells than in exponentially growing cells (Fig. 1-8B), suggesting that PtCdk2 activity is associated with cell proliferation. 1.2.6 PtCdk2 histone HI kinase activity peaks at the end of the cell cycle. 65 Since PtCdk2 appears to be a protein kinase based on its primary sequence, I tested the kinase activity associated with PtCdk2. As PtCdkl has previously been shown to be able to phosphorylate bovine histone HI in vitro (Tang et al., 1997), histone HI was chosen as a substrate for testing PtCdk2 kinase activity. As expected, immunoprecipitates by anti-PtCdk2 antibody of lysate from exponentially growing cells showed significant bovine histone HI kinase activity whereas immunoprecipitates by pre-immune serum did not (Fig. 1-9: PI lane). To examine PtCdk2 kinase activity as a function of cell cycle stage, immunoprecipitation was performed with anti-PtCdk2 antibody using lysates from synchronized samples obtained by elutriation. As shown in Figure 1-9, the protein kinase activity varied periodically throughout the cell cycle, rising to a peak about 9.5 hours after elutriation (Fig. 1-9A). This peak corresponds to the point at which more than 75% of cells had completed cytokinesis. The protein kinase activity increased to about 10 times that observed during early stages of the cell cycle (Fig. 1-9B). This observation suggested that PtCdk2 function might be associated with cell division or division-associated morphogenesis. However, the actual relationship between these two events requires further detailed investigations. 66 HOURS POST REINOCULATION 0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.511.5 PI < - C d k 2 kDa t 2 47.7 — 34.6 — — — 28.3 — B Fig. 1-8. Expression of PtCdk2 protein. Panel A: PtCdk2 protein level during the vegetative cell cycle. Paramecium lysates were prepared from synchronized samples obtained at 1 h intervals, starting from 0.5 h after centrifugal elutriation. Immunoblotting of equal amount of lysate protein was performed with anti-PtCdk2 antibody. PI was lysate from asynchronous sample probed with preimmune serum as a control. Panel B : PtCdk2 protein level in exponentially growing and starved cells. Immunoblotting of equal amount of protein from exponentially growing cells (lane 1) and starved cells (lane 2) was performed with anti-PtCdk2 antibody. 67 H O U R S P O S T R E I N O C U L A T I O N 0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 PI Histone H1 100 80 + |eo H x < 20 + 100 0 4 3 3 3 3 3-0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 HOURS POST REINOCULATION -Cdk2 H1 activity -% of cell division Fig. 1-9. PtCdk2 exhibits protein kinase activity towards bovine histone HI that fluctuates in a cell cycle-dependent manner. Small early G l cells were selected by centrifugal elutriation and reinoculated into fresh medium, and aliquots were taken for determination of cumulative percentage of cell division (B). Equal amounts of protein lysate from synchronized samples were immunoprecipitated with anti-PtCdk2 antibody. The resulting immunocomplexes were subjected to histone HI kinase assay, and aliquots of reaction were run on a SDS-PAGE (A). Quantitation of the kinase activity was performed by scintillation counting of the excised phosphorylated histone HI bands (B). PI shows the background activity in the immunocomplexes by pre-immune serum. 6 8 1.3 DISCUSSION I used PCR techniques to clone and identify a novel gene, PtCDKl, whose predicted amino acid sequence bears a high degree of similarity with Cdks from other organisms. Like PtCdkl, the first Paramecium Cdk cloned previously (Tang et al., 1995), PtCdk2 sequence has all catalytic domains of protein kinases and contains the conserved Cdk hallmark — 'PSTAIRE' region without alteration. Residues equivalent to the regulatory phosphorylation sites, Thrl4, Tyrl5 and Thrl61 in human CDK1, can be found in the Paramecium sequence at comparable locations, which suggests that regulation of PtCdk2 activity in P. tetraurelia may be similar to human C D K L While both PtCdkl and PtCdk2 display sequence homology to Cdks from other species, ranging from 33 to 61%, they share only 48% homology with each other, suggesting they may have distinct functions in the cell cycle regulation. As comparisons, human CDK1 (M-phase CDK) and CDK2 (S-phase CDK) share 65% identity while CDK1 and CDK6 (Gl -CDK) have 47% identity at amino acid level (Meyerson et al. 1992; Pines 1996). Comparison between Paramecium and human Cdks indicated the closer relationship of human CDK1 with Paramecium PtCdk2 than with PtCdkl . It seems to be in agreement with the role of PtCdk2 in cell division. Previous results suggested the presence of two classes of Cdks in P. tetraurelia that differed from each other in their pl3' v" c 7 binding property (Tang et al., 1994). By comparing PtCdk2 with PtCdkl sequences, I found that PtCdk2 had better conservation of putative pl3sucl binding sites than PtCdkl (Fig. 1-3). Specifically, among 14 residues involved in p l3 s " e ; binding of human CDK1 (Ducommun et al., 1991; Marcote et al. 1993), nine can be found in PtCdk2 whereas only six in PtCdkl. However, pl3™ c ; binding 69 experiment with PtCdk2 specific antibody shows that PtCdk2 in fact does not bind to pl3 4 " e / . Therefore, I postulated that the p35 polypeptide recognized by anti-PSTAIRE antibody might contain two different polypeptides with the same migration rate on SDS-PAGE, but with distinct pl3sucl binding properties. Immunodepletion of pl3sucl bound polypeptide from lysate confirmed that a portion of p35 polypeptide recognized by anti-PSTAIRE antibody did not possess affinity for pl3™' ;, and this might correspond to PtCdk2. That anti-PtCdk2 antibody only recognized this portion of p35, not that bound to pl3'™e/ (PtCdk3) further suggests that the unbound form of p35 is PtCdk2. Identification of PtCdk2 as a new Cdk strengthens the notion that multiple Cdks are present in P. tetraurelia, and that cell cycle regulation in ciliates may be more complicated than was thought before. However, it is not yet known whether these Cdks have overlapping functions or whether each has a distinct stage-specific function in the Paramecium cell cycle. It is interesting that none of the Paramecium cell cycle mutants isolated so far is mutated in CDK, which is likely to be attributable to the presence of isoforms of the Paramecium CDK genes, as suggested by the genomic Southern blotting results and the isolation of a PtCDKl gene isoform in previous study (Tang et al, 1995). As predicted from its primary sequence, PtCdk2 immunoprecipitate exhibits histone HI kinase activity. While PtCdkl kinase activity peaks at the initiation of macronuclear D N A synthesis (Tang et al., 1997), PtCdk2 activity reaches its maximal level when more than 75% of cells have divided. This further distinguishes it from the PtCdk3 kinase activity associated with pl3'™c;. The maximum activity level of PtCdk3 was reached -90 min before cytokinesis (Tang et al., 1994), which coincides temporally with PCD, the 70 major control point in the Paramecium cell cycle. Thus, the profile of PtCdk2 kinase activity suggests that it may have a role in the control of cell division-related processes. Since the abundance of PtCdk2 protein remained relatively constant during the vegetative cell cycle, it is likely that PtCdk2 kinase activity, like most Cdks, is regulated by post-translational modifications, given that both cyclin binding domains and regulatory phosphorylation sites are conserved within the sequence. Consistent with this, two classes mitotic cyclin homologues have been identified in P. tetraurelia (see Chapter 2; Zhang et al., 1999). Results from co-immunoprecipitation experiment indicate that PtCdk2 is in a complex with one of the cyclins (PtCyc2), and that PtCyc2 associated histone HI kinase activity also reached a maximum at the end of the cell cycle. Taken together, the results demonstrate that a family of three Cdk-like protein kinases occurs in P. tetraurelia: PtCdkl, PtCdk2 and PtCdk3. Given that their kinase activities reach maximal levels at different points during the cell cycle, and that the proteins exhibit different affinities for p l3 ' " w beads, it is likely that multiple Cdks evolved to regulate different cell cycle events early in the evolutionary history of ciliates. This view is further supported by the identification of two anti-PSTAIRE reactive proteins from another ciliate, Tetrahymena thermophila, with different affinities for pl3sucl (Roth et al. 1991; Tang et al., 1994; Zhang et al., in preparation; Chapter 3). 71 CHAPTER 2. TWO DISTINCT CLASSES OF MITOTIC CYCLIN HOMOLOGUES, CYC1 AND CYC2, ARE INVOLVED IN C E L L C Y C L E REGULATION IN CILIATE PARAMECIUM TETRAURELIA 2.1 INTRODUCTION Cyclins were initially discovered in the cleaving eggs of marine invertebrates and were named after their pronounced synthesis and accumulation during interphase followed by abrupt destruction at metaphase/anaphase transition (Evans et al., 1983; Standart et al., 1987; Swenson et al., 1986). Cyclins have since been identified in a wide variety of species, and form part of the conserved mechanism of cell cycle regulation. They confer activity on the associated Cdk partners at appropriate times during the cell cycle, and are thought to determine the subcellular localization and substrate specificity of these Cdks (Peeper et al., 1993; Pines and Hunter, 1991). Cyclins are diverse in sequence, sharing homology only over a region of about 100 amino acids that has been designated the 'cyclin box' (Minshull et al., 1989; Pines and Hunter, 1989). It is this region that is mainly involved in binding to the Cdk subunits, although binding is also influenced by regions C-terminal to the cyclin box (Kobayashi et al., 1992; Lees and Harlow, 1993). Previous work has demonstrated the presence of three different Cdks in P. tetraurelia that exhibit activities associated with different cell cycle stages (Tang et al., 1994, 1995, 1997; Zhang and Berger, 1999; Chapter 1). The histone HI kinase activity peak at IDS and the localization of the protein to the macronucleus suggests a role for PtCdkl in macronuclear D N A synthesis. On the other hand, the kinase activity peak of PtCdk3, the pl3™ e i binding Cdk, coincides temporally with the PCD (Tang et al, 1994, 1997). PtCdk2 activity peaks late in the cell cycle (Zhang and Berger, 1999). The 72 regulation of PtCdkl and PtCdk2 kinase activity during the vegetative cell cycle does not involve oscillations in their transcript or protein levels. Major regulatory phosphorylation sites equivalent to Thrl4, Tyrl5 and Thrl61 in human CDK1, and which are shared among most Cdks, are conserved in both PtCdkl and PtCdk2. Regions previously shown to be involved in cyclin binding such as PSTAIRE region in protein kinase domain III and the threonine residue (Thrl61 in human CDK1) in domain VIII (Pines and Hunter, 1990) can also be found in both sequences. It seems possible that the regulation of the kinase activity of the P. tetraurelia Cdk homologues might include binding of cyclins and phosphorylation/dephosphorylation. As Cdks are associated with cyclin regulatory subunits in virtually all other instances, I sought to determine whether cyclin homologues are present in P. tetraurelia, and if so, what roles they play during normal P. tetraurelia cell cycle. Here, I describe the identification of two mitotic cyclin homologues, PtCycl and PtCyc2, from P. tetraurelia, and present evidence for their roles in the vegetative cell cycle regulation. 2.2 RESULTS 2.2.1 Identification of two distinct mitotic cyclin homologues from P. tetraurelia. By comparison of the amino acid sequences of known mitotic cyclins in the respective 'cyclin box' regions, two degenerate primers corresponding to conserved regions within the cyclin box region, MRAILV and ASKYEEI were synthesized and used for PCR with P. tetraurelia genomic DNA as template. Paramecium codon usage was taken into account to limit redundancy (Martindale, 1989). A 198 bp DNA fragment of expected size was consistently obtained, which was then gel purified. After subcloning into 73 pBluescript II KS+ and blue/white selection, 8 positive clones were obtained. D N A sequencing analysis revealed two distinct clones. The derived amino acid sequences of both fragments exhibited extensive homology with known mitotic cyclins. They share about 71% identity at the amino acid level with each other. These two presumptive cyclin sequences were named PtCYCl and PtCYC2, respectively. The PtCYCl D N A fragment was radioactively labeled and used in turn to screen a Xgtl 1 cDNA library derived from mRNA of vegetative cells and a A.EMBL3 genomic D N A library. Restriction mapping and D N A sequence analysis of positive clones isolated from these libraries revealed an open reading frame of 972 bp, expected to encode a protein of 324 amino acids with a predicted molecular mass of approximately 38 kDa. B L A S T search of GenBank showed that the predicted open reading frame exhibited a high degree of homology to known mitotic cyclins. Thus, the protein encoded by PtCYCl was named PtCycl, standing for mitotic cyclin homologue 1 from P. tetraurelia. PtCycl is small compared to mitotic cyclins from other eukaryotes (45-60 kDa), but it displays all the cyclin structural hallmarks (Fig. 2-1 A). Two independent primer extension analyses, using two different antisense primers derived from PtCYCl, demonstrated a further 290 bp from the presumptive initiation codon A U G to the 5' end of transcript. An in-frame stop codon U G A was present upstream of the translational initiation codon A U G , and no other in-frame AUGs upstream in between (Fig. 2-1 A). This corresponds well to the size of the transcript revealed by Northern blot analysis with the PtCYCl probe, in which a single -1.3 kb transcript of PtCYCl was detected in exponentially growing cells, but not in starved cells (data not 74 shown). These results suggest that the PtCYCl sequence obtained is full-length (GenBank accession number AF052484). The full-length cDNA sequence of PtCYCl was obtained by a combination of 5' and 3' R A C E (Frohman et ah, 1990). It contains an open reading frame of 1008 bp and it encodes a protein of 336 amino acids, which is 12 amino acids longer than PtCycl (Fig. 2-1B; GenBank accession number AF052487). Its predicted molecular mass is 40 kDa. PtCyc2 exhibits 48.8% overall identity with PtCycl, and 65% identity within the 'cyclin box' region. Comparison between respective cDNA and genomic D N A sequences revealed that PtCYCl contains two small introns, one is 23 bp and the other 24 bp while PtCYCl has only one intron of 23 bp (Fig. 2-1 A, B). They reside just outside of the 'cyclin box' region. The location of the PtCYCl intron corresponds to that of the second intron in PtCYCl. The abrupt destruction of cyclins at mitosis involves an ubiquitin-dependent proteolysis that requires a short sequence motif, RxxLxxIxN, the so-called 'destruction box' (Glotzer et al., 1991), usually located close to the N-terminus. In PtCycl, the sequence, RCFGKEIAN, corresponding to residues 7-15, bears a single amino acid substitution (Lysine to Glycine) with respect to the destruction box consensus sequence. Similarly, in PtCyc2, R F F G K E L V N , in the corresponding region also shows limited similarity to the destruction box. However, whether they are actually involved in the cyclin destruction in the Paramecium cell cycle needs further investigation. 75 Fig. 2-1. Genomic D N A sequences of P. tetraurelia CYCla (A) and CYC2 (B), and the predicted amino acid sequences of their gene products. 'Cyclin box' regions are single underlined. 'Destruction box' regions close to 'RxxLxxIxN ' consensus sequence are double underlined. Introns are shown in lower case. Potential Cdk phosphorylation sites (Thr 269 and Thr 274 in Cycl A and Thr 270 in Cyc2) are indicated by '#'. Stop codons (TGA) in both sequences are indicated by '*'. 76 A AAAATTATCCTAAT -277 ACTTAATCATATCAAATGAGAAAATAAGAATATCCTTTTACTTTGACTGATTATAGAATTTGCTCAGTA -2 0 8 TATTCAATATTAAAAATAACATTTGATTATATGAATCAGAGTGTTTCTTTGTTTCTACCTTTAATGGTT -13 9 TCTCAATTTCAGATTAAAGAGCGGTAAAATTGAAATAATAAATAAGATAATTAAAAATCCATTTTCACT - 7 0 TTATTTATCACTTTTATATATAAGTGTTTAGTTTTTAACGATAATAATAATTTATTTAATATTAACAGA -1 ATGATCATCGAAAATCAAAGATGTTTTGGGAAGGAAATAGCAAATTCAACTTTGCATCAATCCAAGGAG 6 9 M I I E N Q R T F G K F . T A M S T L H Q S K E 23 ATAGGCATAATCGTGGAAAAACATAAAAAACCTTTCTCCATAATACCAAAAGTTTTTGCAATGAGTTTG 138 I G I I V E K H K K P F S I I P K V F A M S L 46 GATGATAAAGAAAATAAACTGTTTAGAAGAGAATCAGAAAAATTCTAAATTGAGATAGAAACCGAAAAG 2 07 D D K E N K L F R R E S E K F Q I E I E T E K 69 AGCAAGGATGTTAAAAATCCTTAAAATGTTGAGTTATATTCCAATGAGATCTTACAACACCTACTGATT 27 6 S K D V K N P Q N V R L Y S N E I L Q H L L I 92 GAGGAGgtaatatttttagactattt aagAATAAATATACAATTAACTAGTACATGACACCTGAGTAA 344 E E N K Y T I N Q Y M T P E Q 107 CAGCCTGATATCAACAT AAAAATGAGAGCTATTCTTGTGGATTGGTTAATTGATGTTCATGCCAAATTC 413 Q P D I N I K M R A I L V D W L I D V H A K F 130 GAGTTGAAGGATGAAACACTCTACATTACAATCTCTTTGATTGATCGATACTTGGCTCTGGCTCAAGTC 4 82 E L K D E T L Y I T I S L I D R Y L A L A O V 153 ACAAGAATGAGATTACAGTTAGTAGGTGTGGCTGCCCTGTTTATAGCTTGTAAGTACGAAGAAATCTAC 551 T R M R L O L V G V A A L F I A C K Y E E I Y 176 CCTCCTGCTTTGAAGGATTTCGTTTACATAACAGATAATGCGTATGTGAAAAGTGACGTTTTGGAAATG 62 0 P P A L K D F V Y I T D N A Y V K S D V L E M 199 GAAGGTTTAATGTTATAAGCCTTAAATTTCAATATATGCAATCCCACTGCTTATTAATTCTTACAAAAA 689 E G L M L O A L N F N I C N P T A Y O F L O K 222 TACTCAACCAATTTAGATCCGAAGGATAAGGCGTTAGCTTAATATATACTGGAATTGGCTTTAGTTGAA 758 Y S T N L D P K D K A L A O Y I L E L V L V E 245 TAT AAATTT ATTATATATAAGCCTTCTT AAATTGTCC AATCTGTT ATATTTTTAGTT AATAAGATT AGg 827 Y K F I I Y K P S O I V O S V I F L V N K I R 268 taatacttttatattaatcatagAACACCCACTTATAAAACACCGAACGAGAATCAATTAAAGCCTTGT 896 T # P T Y K T # P N E N Q L K P C 283 GCTAAAGAATTATGCACATTACTTTAAACAGCAGATCTAAGTTCCCTATAAGCAGTAAGGAAGAAATTC 965 A K E L C T L L Q T A D L S S L Q A V R K K F 306 AATGCTTCTAAATTTTTTGAAGTTTCGAGAATTAAAGTAGAAAAAACAAACAAATGATTGTAGTTTTAA 1034 N A S K F F E V S R I K V E K T N K * 324 ATGTCTTGTTTCTTTGACTTTCACTCAAATTTTCCA 107 0 B GATATATTTTAAC AGAATATTATATTTTTTACGTATAAT AAA -1 ATGATCCACGAACAGTATAGATTCTTCGGCAAAGAATTGGTTAATTCATCGGGTGAATTCTAAAAGGAA 69 M I H E Q Y R F F G K K T , V N S S G E F Q K E 23 AACACAGACAGAAATAAACCAAAGCATCTTACTGTATTACCTAGATATCTCAAACTAAATTTATGGTAG 13 8 N T D R N K P K H L T V L P R Y L K L N L W Q 46 GAAG AGAAAGAAAATAAAATGGAAGTTGAAAATAATACTCAACAATTATGTTCTTTTGATTAACAAATG 207 E E K E N K M E V E N N T Q Q L C S F D Q Q M 69 ATTAAAGACCCTTAATACACTTCTTTATATAATAAGGAGATCTATACTTATTTGTTGACTTAAGAAGAA 27 6 I K D P Q Y T S L Y N K E I Y T Y L L T Q E E 92 AAATATTTAGTTAGTAATAATTACATGAATGAATAATAACAACCTGACTTAAATGCAAGAATGAGAGCA 345 K Y L V S N N I M N E Q Q Q P D L N A R M R A 115 ATTCTCTTAGATTGGTTGATTGATGTCCATCTCAAATTTAAATTAAGAGATGAGACCTTATATGTTACA 414 I L L D W L I D V H L K F K L R D E T L Y V T 13 8 ACTTATTT AATTGACAGGTTTCTTAATTTT AAG ACT AC AACC AG AT AAT AACTTTAATT AGTTGGAGT A 483 T Y L I D R F L N F K T T T R O O L O L V G V 161 GCTTC ATT ATTTATAGCTTGT AAAT ATGAAGAAAT AT ATCCCCCTG ATTTGAAAGATTTTGTGTAC ATC 552 A S L F T A C K Y E E I Y P P D L K D F V Y I 184 ACTGAC AATGCCTACAC AAAAT AGG ATGTCCTTGAAATGGAAGGAT AAAT ATTATAAACATTAGACTTC 621 T D N A Y T K O D V L E M E G O I L O T L D F 2 07 TCCATAACCCAACCATCAAGTTATTGTTTTCTTTAGAGATTTGGTAGAATTGCAGGATTAGATACTAAG 690 S I T O P S S Y C F L O R F G R I A G L D T K 23 0 AATTTAAGCTTGGCTTAGTATCTTTTAGAGCTATCAATTGTTGACATCAAATTTATGAACTATAAACCA 759 N L S L A O Y L L E L S I V D I K F M N Y K P 253 TCGTTTCTATCTGCAGCAGCTATTTATTTAGTCCACAAAATAAGgtatcagaaatatattttcatagG 827 S F L S A A A I Y L V H K I R 268 AAGACTCCTC AATCTTGGAGTG AAGAAATGC AGAAAATG ACTGGTTAT AATGAATAAGAACTT AGGTAT 896 K T # P Q S W S E E M Q K M T G Y N E Q E L R Y 291 TGTGCTAAAGAAATGTGTCTTGTTTTGCAATCATCAGACAAATCAAATTTATAGGCTGTTAGAAAAAAG 965 C A K E M C L V L Q S S D K S N L Q A V R K K 314 TTTGCTT AGCCTAAATACT AAGAAGT ATCACGTATTAGAGTAGAAAGGC AAATTAAATAATAAAAATG A 1034 F A Q P K Y Q E V S R I R V E R Q I K Q Q K * 336 ATGGTTAATTCATTTCTAGTTAAAATGTTTAACATCTATAAATTTATYATCAATTTTTTTAA 1096 77 2.2.2 Genomic organization of the P. tetraurelia cyclin genes. As an independent pursuit of the complete sequence of PtCYCl, 5' and 3' RACEs were carried out. D N A sequence analyses of RACE-PCR products revealed another sequence which displayed overall 90.4% identity at the amino acid level and 85.9% identity at the nucleotide level with PtCYCl that was cloned by the library screening approach. Of 123 altered codons, 92 were silent changes and 31 resulted in amino acid changes, of which 13 were conservative substitutions (GenBank accession number AF052486; Appendix II). To distinguish them, I designated the gene cloned from the libraries as PtCYCla and this second gene as PtCYClb. The corresponding protein products were named PtCycl A and PtCyclB, respectively. A Southern blot analysis of genomic D N A digested with five restriction enzymes, EcoRI, Hindlll, Pstl, Xbal and BamHI, was carried out with PtCYCla and PtCYClb probes from the cyclin box region under high- and low-stringency conditions (Fig. 2-2). No recognition sites for any of these enzymes were found in the sequences. Single hybridizing bands were detected in all five restriction digests under high-stringency conditions (Fig. 2-2B, C). Under low-stringency conditions, on the other hand, two bands were visible in each lane. The weaker hybridizing bands detected with PtCYCla under low-stringency conditions corresponded to the bands detected with PtCYClb under high-stringency conditions (Fig. 2-2A), and vice versa (data not shown). Therefore, P. tetraurelia appears to carry one copy for each of the PtCYCla and PtCYClb genes. When the blot was stripped and re-probed with the PtCYC2 probe corresponding to the cyclin box region, single hybridizing bands were detected in each lane under both high-and low-stringency conditions (Fig. 2-2D). I concluded that the PtCYC2 gene is also 78 present as single copy genes in the Paramecium genome. Moreover, the discrete patterns of hybridization for PtCYCla, CYC lb and CYC2 indicate that they are not tandemly arranged in the genome. As PtCYCla and PtCYClb have the same 5' and 3' flanking sequences and a high degree of homology, it is likely that they represent two isoforms of the same gene. Similarly, PtCDKl from P. tetraurelia also has two closely related isoforms (Tang et al., 1995). No other hybridizing bands were observed with PtCYCla, PtCYClb and PtCYC2 probes when washes were carried out at 37°C (data not shown), indicating the absence of additional cyclins with strong cyclin box sequence similarity with PtCYCl and/or PtCYC2. These results, however, do not exclude the possibility that there are other cyclin families in P. tetraurelia that can not be detected by hybridization to these probes. 2.2.3 Paramecium mitotic cyclins do not belong to either A- or B-type cyclins GenBank search has shown that the "cyclin box" sequences of the Paramecium PtCycl and PtCyc2 proteins are more homologous to those of mitotic cyclins than to those of G l cyclins. Furthermore, sequences resembling the "destruction box" consensus can be found in the N-termini of both cyclins. Therefore, PtCycl and PtCyc2 are clearly homologues of mitotic cyclins. Appendix III shows an alignment of PtCycl and PtCyc2 sequences with A- and B-type cyclins from other eukaryotes in the cyclin box region. The Paramecium cyclins show significant homologies to both A and B cyclins but do not appear more closely related to either type in terms of overall amino acid identity in this region. They both exhibit 42— 51% identities with both A- and B-type cyclins. Nor is it easy to assign the Paramecium cyclins to either A or B types by using sequence motif. The PtCycl sequence differs in 79 19/70 positions from the consensus established previously for A-type cyclins, as opposed to 25/69 positions from the consensus for B-type cyclins (Hata et al., 1991). As for PtCyc2, it differs in 21/70 positions from A-type cyclin consensus sequence whereas 24/69 from B-type cyclin consensus sequences. None of the Paramecium cyclins has the E V X E E Y K L motif (amino acids 11-18 of the cyclin box) found in all A-type cyclins. Similarly the F L R R X S K motif that is found in all B-type cyclins (amino acids 105-111 of the cyclin box) is not conserved in any the Paramecium cyclins. Therefore, PtCycl and PtCyc2 are almost equally related to cyclins of the types A and B in the cyclin box region. A phyletic tree was constructed based on the sequence alignment, as shown in Figure 2-3. While A- and B-type cyclins from other eukaryotes form their respective groups as expected, the Paramecium cyclins do not fit into either A or B-type mitotic cyclins and constitute a separate group. Therefore, PtCycl and PtCyc2 may represent a novel and distinct type of cyclin. 2.2.4 PtCycl and PtCyc2 protein levels display distinct cell cycle-dependent fluctuations in the vegetative cell cycle. To characterize the cyclin gene products, antisera were prepared against PtCycl A and PtCyc2, respectively. The sequence similarity of the PtCycl A and PtCyclB proteins is sufficiently great that it is very unlikely that they could be distinguished by the polyclonal antibody I made here. Therefore, the antibody against PtCycl A has been denoted as anti-PtCycl antibody. On an immunoblot of total lysate from exponentially growing P. tetraurelia cells, anti-PtCycl antibody recognized a protein of apparent molecular mass of 38 kDa, while anti-PtCyc2 recognized a protein of 40 kDa. Both their sizes agreed with the predicted molecular masses determined from respective primary sequences. These 80 proteins were not detected when pre-immune serum was used (Fig. 2-4A and B, PI lanes). The immunoreactivities were specific to their cognate antigens, since preincubation of the immune sera with antigens at 4°C for 3.5 h abolished the appearance of the bands (data not shown). Sometimes, anti-PtCyc2 serum also detected a faint band of 45 kDa on the western blot (Fig. 2-6B, lysate lane), however, this band did not exhibit any cell cycle-dependent fluctuation in its abundance, indicating that it may be due to a non-specific cross-reaction of the anti-peptide antibody. To determine whether PtCycl and PtCyc2 display a periodic pattern of synthesis and destruction during the vegetative cell cycle, P. tetraurelia cells were synchronized by centrifugal elutriation. The median time of cell division in elutriation sample ranged from 8.5 to 9.5 h post elutriation. Protein samples were prepared at 1 h intervals and subjected to immunoblot analysis. As shown in Figure 2-4, both PtCycl and PtCyc2 protein levels varied during the cell cycle. PtCycl level was low immediately after elutriation, began to increase at about 5.5 h, reached a peak at -7.5 h, and then decreased gradually upon the onset of cytokinesis (Fig. 2-4A). The timing of the peak, observed at 1-2 h before most cells entered cytokinesis, coincides with the PCD. On the other hand, PtCyc2 was almost not detectable until 6.5 h after elutriation. The maximal level of PtCyc2 expression was found late in the cell cycle after most of cells had completed cytokinesis (Fig. 2-4B). By this time, PtCycl level has declined. By the time that all cells finished division, the PtCyc2 protein level had dropped to an almost undetectable level. As expected, both PtCycl and PtCyc2 protein levels were dramatically reduced in starved cells (Fig. 2-5), suggesting that the roles of PtCycl and PtCyc2 are associated with cell proliferation. 81 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 M (kb) 1 2 -i o - tp w W w « * * * • 7— 4 — 3 — 2 -1.6 1 — H i n MM Si-' ^HB ' . .-i. . i^fSRx , - ; . ' L - Y B C D Fig. 2-2. Genomic Southern blot analysis of Paramecium genomic D N A with probes derived from PtCYCla, CYClb, and CYC2, respectively. Genomic D N A was digested with EcoRI (lane 1), Hindlll (lane 2), Pstl (lane 3), Xbal (lane 4), and BamHI (lane 5), then probed with DIG-labeled CYCla D N A probe under low stringency conditions (A). The membrane was then stripped and reprobed under high stringency conditions with PtCYCla (B), PtCYClb (C), and PtCYC2 (D) probes sequentially. The 1-kb marker from Gibco-BRL is indicated on the left. 82 — Paramecium PtCyc2 — Paramecium PtCycl B — Paramecium PtCyd A Drosophila DmCYCA — I Spisula SsCYCA I Xenopus XICYCA1 : Homo HsCYCA i Spisula SsCYCB I Xenopus XICYCB1 Homo HsCYCBI Dictyostelium DdCYCB Drosophila DmCYCB Fig. 2-3. Evolutionary tree of cyclins from P. tetraurelia (Pt), Spisula solidissima (Ss) (Swenson et al., 1986; Westendorf et al, 1989), Drosophila melanogaster (Dm) (Lehner and O'Farrell 1989; 1990), Xenopus laevis (XI) (Minshull et ai, 1989; 1990), Homo sapiens (Hs) (Pines and Hunter 1989; Wang et al. 1990), and Dictyostelium discoideum (Dd) (Luo et al., 1994). It was based on the alignment of whole cyclin box (see Appendix III). The scale bar represents 0.1 nucleotide substitutions per site. 83 Hours Post Re-inoculation 0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.511.5 PI A ' * •• #•»*• «—» «*•* *~ ** ""**"* ~ PtCycl B *" - PtCyc2 Fig. 2-4. Oscillation of PtCycl and PtCyc2 protein levels during the vegetative cell cycle of P. tetraurelia. Small cells in early G l were selected by centrifugal elutriation and reinoculated into fresh medium. After 0.5 h, aliquots were taken at 1 h intervals to determine protein levels by Western blot analysis using anti-PtCycl and anti-PtCyc2 antibodies. PI: preimmun serum controls. PtCycl PtCyc2 A B Fig. 2-5. Western blot analysis of protein levels of PtCycl (A) and PtCyc2 (B) in P. tetraurelia cells that were exponentially growing or starved. Equal amounts of protein were loaded on each lane. 84 2.2.5 PtCycl and PtCyc2 associate with different Cdks in P. tetraurelia. To determine whether a physical interaction occurred between P. tetraurelia cyclins and Cdks, I examined the ability of PtCycl and PtCyc2 antibodies to coirnrnunoprecipitate Cdks. Both PtCycl and PtCyc2 immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblot analysis for the presence of PtCdkl and PtCdk2 with respective antibody. I found that PtCdk2, but not PtCdkl coprecipitated with PtCyc2 (Fig. 2-6A), and that neither PtCdkl nor PtCdk2 coprecipitated with PtCycl. However, when the same blot was probed with anti-PSTAIRE antibody, a protein of -34 kDa was identified in the PtCycl immunoprecipitate, suggesting that PtCycl associates with a Cdk other than PtCdkl and PtCdk2 (data not shown). Furthermore, a cell cycle-dependent histone HI kinase activity was detected in the PtCycl immunoprecipitate, confirming that a Cdk was associated with PtCycl (see below). The converse experiment was also carried out to confirm the association of PtCyc2 with PtCdk2. PtCdk2 immunoprecipitate were probed with PtCycl and PtCyc2 antibodies, only PtCyc2, not PtCycl, was detected (Fig. 2-6B). As maximal expression of PtCycl was observed at around PCD, and the histone HI kinase activity associated with PtCdk3 also peaks at PCD, I suspected that PtCycl might be associated with PtCdk3, as PtCdk3 is the only known Cdk that can bind to yeast p l3 i " c 7 in P. tetraurelia, If it is true, I predict that the PtCycl protein should be precipitated by pl3™c 7 beads. As expected, I found that PtCycl was present in the p l3 O T e / bound fraction (Fig. 2-6C). Hence, it is inferred that PtCycl associates with pl3™ c 7 by virtue of its association with PtCdk3. However, without a specific antibody against PtCdk3, direct evidence is not yet available to support this possibility. 85 2.2.6' PtCycl and PtCycl form active histone HI kinases with respective Cdk partners. Since all three Cdks in P. tetraurelia have been demonstrated in the previous studies to have histone HI kinase activity (Tang et al., 1994, 1997; Zhang and Berger, 1999), I assayed PtCycl and PtCyc2 immunoprecipitates for histone HI kinase activity to determine if cyclin forms active complexes with Cdk in P. tetraurelia. Both PtCycl and PtCyc2 immunoprecipitates displayed kinase activity towards bovine histone HI , whereas immunoprecipitates using preimmune sera did not have such activity (Fig. 2-7). Since PtCycl and PtCyc2 do not contain any known protein kinase sequences, it is almost certain that associated Cdk homologues are the kinases (Hanks et al., 1988). To determine if the histone HI kinase activities in the cyclin immunoprecipitates were cell cycle-dependent, PtCycl and PtCyc2 immunoprecipitates prepared from lysates of synchronous cells by elutriation were assayed for histone HI kinase activity as a function of stage in the cell cycle. As expected, based on the periodicities of the PtCycl and PtCyc2 protein amounts, a kinase activity peak for the PtCycl immunoprecipitate was observed at approximately the PCD in the elutriated cell population (Fig. 2-7A), while the PtCyc2 associated kinase peak was at the end of the cell cycle during cytokinesis (Fig. 2-7B). The PtCycl immunoprecipitate showed a peak of kinase activity about 2 h earlier than that of the PtCyc2 immunoprecipitate. The PtCycl associated kinase activity had fallen to 15% of its maximal level by the time the kinase activity of PtCyc2 immunoprecipitates reached its peak. These results suggest that the Paramecium cyclins can form active complexes with Cdks and distinct Cdk/cyclin complexes may fulfill different functions in cell cycle regulation. 86 t i CP CJ J $ k D a J # kDa P S 4 7 ' 7 ~ m PtCycl 34.6 — ^ -4- PtCyc2 — 47.7 — P t C d k 2 - * - 3 4 . 6 - „ 2 8 . 3 -— 28.3 — B Fig. 2-6. Interaction between Cdk and cyclin proteins in P. tetraurelia. More than 2.5 mg of total proteins from exponentially growing Paramecium cells was used in each co-immunoprecipitation experiment. Interaction between PtCyc2 and PtCdk2 was demonstrated by reciprocal coimmunoprecipitation experiments. PtCdk2 was detectable in the PtCyc2 immunoprecipitates (A) while PtCyc2 was detectable in the PtCdk2 immunoprecipitates (B). * indicates a non-specific band detected by anti-PtCyc2 peptide antibody. The heavy band above 47.7 kDa represents rabbit IgG. PtCycl was detected in p i y * -bound fraction (Pellet, P lane), but not in unbound fraction (Supernatant, S lane), by anti-PtCycl antibody (C). Bands below 34 kDa might be attributed to degraded products of the proteins of interest. 87 2.2.7 Conservation of cyclin-like sequences in ciliates Blepharisma intermedium, Sterkiella histriomuscorum, Colpoda sp. and Tetrahymena thermophila were chosen as representatives of Heterotrichia, Spirotrichea, Colpodea and Oligohymenophorea, respectively. By utilizing the degenerate primer pair used for cloning the P. tetraurelia cyclins, PCR was carried with genomic DNAs of these ciliates as templates. PCR products of expected size (180 bp) were gel purified and subcloned into pBluescript II KS +. D N A sequencing analysis of resulting clones revealed that one class each from B. intermedium, S. histriomuscorum, C. sp. and T. thermophila exhibited high degree of homology to mitotic cyclins from both P. tetraurelia and higher eukaryotes, suggesting that they were all homologues of mitotic cyclins (Fig. 2-8; GenBank accession numbers: TtCyc, AF165220; BiCyc l , AF165222; ShCyc, AF165223; CsCyc, AF165224). Another cyclin-like sequence with an internal U G A stop codon was also identified from B. intermedium, presumably representing a pseudogene (BiCyc2, AF165221). Within this conserved region within cyclin box, the amino acid identity shared by the cyclin-like sequences from B. intermedium, S. histriomuscorum, C. sp., T. thermophila and P. tetraurelia range from 59 to 98%. 88 Fig. 2-7. Oscillation of PtCycl and PtCyc2 associated histone HI kinase activity during the vegetative cell cycle of P. tetraurelia. Small cells in early G l were selected by centrifugal elutriation and reinoculated into fresh medium. After 0.5 h, aliquots were taken at 1 h intervals to determine cumulative percentage of cell division; histone HI kinase activity present in PtCycl (A) or PtCyc2 immunoprecipitates (B). Aliquots of kinase assay reaction were run on SDS-PAGE, and dried gels were exposed to X-ray films (the upper panels in A and B). Protein levels in Fig. 2-4 were quantitated using the NIH Image program, and quantitation of the kinase activities was carried out by scintillation counting of the excised phosphorylated histone HI bands. Results are shown as percentage of maximum (the lower panels in A and B) (Note: this figure presents composite results derived from different batches of synchronized samples due to the limited amount of protein available from each elutriation. 89 Hours Post Re- inoculat ion 0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 PI m m .mm... '>M • *- - Histone H1 X < H O U R S P O S T R E I N O C U L A T I O N - C y c l protein — • — C y c l assoc ia ted kinase - -*- - % of cel l division B Hours Post Re-inoculation 0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 PI H P ™ Histone H1 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 H O U R S P O S T R E I N O C U L A T I O N - C y c 2 protein - C y c 2 associated kinase •tr - % of cel l division 90 P t C y c l A MRAILVDWLIDVHAKFELKDETLYITISLIDRYLALAQVTRMRLQLVGVAALFIACKYEE P t C y c l B - - - — V - - - K - S K P t C y c 2 — L- - L - - K - - R - - -— - V - - T Y — — F - N F K T T - -QQ S T t C y c 1- - - I V E I - E - CK-- L P - - - - F - - - V N - - — F - D R - T C - - D N — T - S - -ShCyc E - - L - -R--QR-- - V K I - - L — E K Q M - -KS - - T S - L - - S - -B i C y c 1- - C I - S - - F - - H - -QP--- - F L - - V N - - E R S - I A K E N — T - S - -C s C y c 1- - C I - S - - F - - H - -QP--- - F L - - V N - - E R S - I A K E N - P - - — T - S - -Fig. 2-8. Alignment of cyclin-like sequences from B. intermedium, S. histriomuscorum, C. sp. and T. thermophila with corresponding cyclin box region of PtCyclA, B and PtCyc2 from P. tetraurelia. Dashes indicate identity with PtCyclA. 91 2.3 DISCUSSION I have shown that cyclins, the activating subunits of Cdk kinase activity, are also present in P. tetraurelia. This is the first time that cyclin homologues have been unambiguously identified, cloned, and characterized in ciliates, although a protein that exhibited a cell cycle-dependent oscillation in abundance was observed in Tetrahymena thermophila (Williams and Macey 1991), and a sequence that displayed a hmited degree of homology to B-type cyclins was reported earlier in Stylonychia lemnae (Maercker and Lipps 1994). My study not only extends the omnipresence of cyclins to this unique and diverse, unicellular group, but also lays the foundations for further investigation of the molecular basis of the unusual cell cycle control system in ciliates. The assignment of PtCycl and PtCyc2 as P. tetraurelia mitotic cyclins is based upon their sequence similarity to other mitotic cyclins and their cell cycle-dependent oscillation at both protein level and associated histone HI kinase activity. The sequence similarity is restricted to the cyclin box regions, and to the ubiquitin specific 'destruction box' sequences near the N-termini. Like most mitotic cyclins examined thus far, both PtCycl and PtCyc2 display periodic oscillation in their protein levels during the vegetative cell cycle, but with different kinetics. PtCycl was maximally expressed at around PCD, about 90 min before cytokinesis, whereas PtCyc2 was maximally expressed at the end of the cell cycle. This difference in periodicity implies that PtCycl and PtCyc2 may have different functions during the Paramecium cell cycle. The presence of multiple cyclins has been well documented in higher eukaryotes. Human cells have at least 16 cyclins with 9 partner kinases (Johnson and Walker, 1999; 92 Pines 1996), and each cyclin appears to bind to specific kinase partner(s), resulting in cyclin/Cdk complexes that play distinct roles during the cell cycle. This also seems to be the case for P. tetraurelia. Reciprocal immunoprecipitation experiments with anti-PtCdk2 and anti-PtCyc2 antibodies demonstrated that PtCyc2 was physically associated with PtCdk2 in vivo, consistent with the coincidence of PtCyc2 protein maximal expression and PtCdk2 associated histone HI kinase activity at the end of the cell cycle. The binding of PtCycl to pl3'™e/ raised the possibility that PtCycl was in association with PtCdk3, which has an activity peak at PCD and is the only known Cdk in P. tetraurelia that binds to pl3'™c/. However, further work is required to clarify this point. As predicted on the basis of the physical association between Cdks and cyclins in P. tetraurelia, the immunoprecipiates prepared by antisera against both cyclins displayed protein kinase activity towards histone HI . Moreover, cyclin-associated histone HI kinase activities showed fluctuations during the vegetative cell cycle: PtCycl-associated histone HI kinase peaked at PCD whereas PtCyc2-associated kinase activity reached the maximum at the end of the cell cycle. Coincidence of the cyclin associated kinase activity with previously described Cdk histone HI kinase activity further confirmed the immunoprecipitation results with antisera and pl3™ c 7, that is, PtCyc2 binds to PtCdk2 whereas PtCycl binds to PtCdk3. As there is no specific antibody against PtCdk3, it was not possible to verify the physical interaction between PtCdk3 and PtCycl. I cannot exclude the possibility that PtCycl forms a complex with an unidentified Cdk that also associates with pl3 ' s u c / and which has an activity peak at PCD. However, the parallel oscillation of the cyclin protein 93 levels with the kinase activity of their respective Cdk partners indicates that PtCycl and PtCyc2 function as Paramecium Cdk activators. Histone HI kinase activities of the resulting cyclin/Cdk complexes reach maxima at different stages of the cell cycle, suggesting functional separation between the Paramecium cyclins. Given the unique cellular structure of ciliates, with temporally and spatially different regulated cell cycle events in macronucleus, micronucleus, and cytoplasm, it is perhaps not surprising to find the presence of multiple Cdk/cyclin combinations with distinct functions in P. tetraurelia. Thus far, a cyclin that associates with PtCdkl, the kinase associated with macronuclear D N A synthesis, has not been identified. Previous evidence from glycerol density gradient centrifugation experiment indicated that PtCdkl might function as a monomer (Tang et al., 1997). Further study is necessary to clarify whether this kinase complexes with a cyclin that has very limited sequence identity to the primers that were used to isolate PtCycl and PtCyc2 or whether it does, in fact, function without a cyclin. It is known that human G l cyclins exhibit great divergence from mitotic cyclins in the cyclin box, and the identity between different classes of human cyclins can be as low as 18% (Cyclin C vs. Cyclin A) (Lew et al., 1991). It is therefore not surprising that the primers used to clone PtCYCl and PtCYCl did not recognize PtCdkl-binding cyclin, given that the function of PtCdkl is more likely to be Gl-related. In spite of small size of the PCR region used for amplifying cyclin-like sequences of other ciliates, it is the most conserved stretch within the 'cyclin box', and all the important residues that are involved in the interaction with Cdk subunits as suggested by crystal structural studies on the CyclinA/Cdk2 complexes can be located within this region 94 (De Bond et al., 1993; Jeffery et al., 1995). Database searches indicated that they showed homology with nothing but mitotic cyclins. Therefore, it seems reasonable for us to conclude that cyclin-like sequences are conserved across major groups within this phylum, despite great diversity suggested by phylogenetic studies. 95 CHAPTER 3. FUNTIONAL CHARACTERIZATION OF A CYCLIN-DEPENDENT PROTEIN KINASE HOMOLOGUE, TtCdkl, IN ANOTHER HOLOTRICHOUS CILIATE TETRAHYMENA THERMOPHILA 3.1 INTRODUCTION In spite of the accumulating molecular and biochemical evidence, presented in previous two chapters, suggesting the presence of Cdk and cyclin homologues and their association with major cell cycle events in P. tetraurelia, in-depth functional characterization of the physiological roles of these Cdk and cyclin homologues in P. tetraurelia is difficult. This is mainly due to the difficulty in D N A transformation as a result of random integration and/or episomal propagation of transforming D N A and the lack of well-characterized cell cycle mutants. In contrast, targeted gene disruption techniques are now available in another holotrichous ciliate, Tetrahymena thermophila (Cassidy-Hanley et al., 1997; Gaertig et al., 1994; Tondravi and Yao, 1986). This organism has a high level of inherent homologous recombination in the macronucleus that permits replacement of the endogenous copies of genes. Moreover, phenotypic assortment (Sonneborn, 1974), a unique feature of T. thermophila, allows the relative abundance of macronuclear D N A molecules carrying the transgene or its endogenous allele to be shifted by selection for or against the paromomycin resistance marker carried in the knockout cassette. Ultimately, with prolonged selection, the endogenous alleles in the polygenomic macronucleus can be completely replaced if they are not essential. Even though T. thermophila and P. tetraurelia are placed in the order Oligohymenophorea that represents a monophyletic group of organisms (Harnmerschrnidt et al., 1996), the evolutionary distance between P. tetraurelia and T. thermophila is far greater than that between rat and brine shrimp, based on molecular phylogeny studies on small subunit 96 ribosomal R N A (ssrRNA) gene sequences (Frankel, 1999; Greenwood et al., 1991). Therefore, by exploring the molecular basis of cell cycle control in T. thermophila, I can determine to what degree information that has been gathered with P. tetraurelia in previous two chapters can be applied to other ciliates, and can be a further step toward understanding the physiological roles that these conserved elements play in ciliate cell cycle regulation. In the study described in this chapter, I isolated the TtCDKl gene and determined the subcelluar localization of its encoded protein. I also partially replaced the endogenous copies of the TtCDKl gene in the somatic macronucleus with a knockout construct containing a paromomycin selection cassette and demonstrated various abnormalities associated with reduced expression of the TtCDKl gene. 3.2 R E S U L T S 3.2.1 TtCDKl encodes a homologue of cyclin-dependent kinases. I used a combination of degenerate and anchored PCR approaches, similar to those used in cloning PtCDKl and cyclins, to isolate a cDNA clone of 1156 bp that encoded a product with a high degree identity at the amino acid level to that of Cdks from other eukaryotes, including P. tetraurelia (Table 4). The largest open reading frame (ORF), starting from the first A T G codon, encodes a protein of 318 amino acids with a predicted molecular mass of -37 kDa (GenBank accession number AF157636, Fig. 3-1). The gene was designated T. thermophila CDK1 (TtCDKl) and its predicted protein product TtCdkl. While it contains the 11 catalytic domains characteristic of protein kinases of all types (Hanks et al., 1988) and shows conservation of all of the regulatory phosphorylation sites in corresponding locations to other Cdks, the 'PSTAIRE' region in TtCdkl has two conservative substitutions out of 16 amino acids, including Ile56-Leu and Leu61-Ile. 97 Table 4. Percentage of amino acid identity between ciliate Cdks and Cdks from other eukaryotes. T. thermophila P. tetraurelia P. tetraurelia Cdkl Cdkl Cdk2 H. sapiens CDK1 53% 55% 58% Z. mays Cdc2ZmA 55% 59% 61% S. pombe Cdc2 51% 53% 58% S. cerevisiae CDC28 53% 56% 57% D. discoideum Cdc2 52% 55% 56% P. falciparum Cdc2 51% 60% 51% E. histolytica Cdc2 47% 46% 45% T. vaginalis Cdc2 37% 41% 33% P. tetraurelia Cdkl 46% 100% 48% P. tetraurelia Cdk2 56% 48% 100% T. thermophila Cdkl 100% 46% 56% 98 TAAAGATAAAAGAGTATCAACTCTCATAAGAAAAAT -7 0 TATTGCTATCAAATATTCGAATTTAAGCAAATAAATCAATCAATCAGTCAGTCATACATACATAATAGG -1 ATGAACAATAATACATTTGGCTCAGACAGATATGAAAAGCTACTTAAAGTAGGTGAAGGAACATATGGT 6 9 M N N N T F G S D R Y E K L L K V G E G T r G 2 3 GAAGTTTACAAGGCTAAAGACATTCAAAGCTCTGAAATTGTTGCTTTGAAGAAGATTAAATTAGAAAAT 13 8 E V Y K A K D I Q S S E I V A L K K I K L E N 4 6 GAAGATGAAGGTGTCCCTTCAACTGCTCTAAGAGAAATTTCAATACTTAAAGAGTTACAACCACATCCC 207 E D E G V P S T A L R E I S I L K E L Q P H P 69 AACATCGTTTGCATGCATGAAGTTATTTATCAACCCTAAGAGAAGAAGTTATACCTTGTATTTGAATTT 27 6 N I V C M H E V I Y Q P Q E K K L Y L V F E F 92 GTTGACTAAGACCTTAAAAAATTCTTAGACTAATACCGCAAAGATAAAAAACTTTAACTTCGTCCTTAC 345 V D Q D L K K F L D Q Y R K D K K L Q L R P Y 115 CAAATCAAACTCATGATGTATCAAATTTTGAATGGATTGAATTTCTGCCACAGCCGTAGAATTATTCAT 414 Q I K L M M Y Q I L N G L N F C H S R R I I H 138 AGAGATTTAAAACCCTAAAACATTTTAATTGATGCTAAGGGAAATATTAAGATTGCTGATTTTGGTTTG 483 R D L K P Q N I L I D A K G N I K I A D F G L 161 GCTAGAGCATTTGGTGTTCCTATCAAGACTCTTACTCATGAAGTTGAAACTCTTTGGTACAGAGCTCCC 552 A R A F G V P I K T L T H E V E T L W Y R A P 184 GAAATTCTTTTAGGCTAAAAGGCTTATAGTCTCGGAGTTGATATTTGGAGTTTGGGTTGCATCTTCCAC 621 E I L L G Q K A Y S L G V D I W S L G C I F H 207 GAAATGGTTGAAAAGAGAGCTCTCTTCATGGGTGACTCTGAAATTGATTAAATATTTAAAATATTCTAA 690 E M V E K R A L F M G D S E ' l D Q I F K I F Q 230 TACCATGGTACTCCTACCGAGTAAACATGGCCTGCTTTGAAGGAATGTCCTTACTTTAAACCTATATAT 759 Y H G T P T E Q T W P A L K E C P Y F K P I Y 253 CCTCGTTTCAAAACTGCTGATCCTAAGACTTACTTTAAGAATTTCTGCGATAAAGGATTTGACCTTATT 82 8 P R F K T A D P K T Y F K N F C D K G F D L I 276 TAATAGATGATTGCTCTTGACCCTGCTAAAAGAATATCAGTAAAGGATGCTCTCAGACATCCCTACTTT 897 Q Q M I A L D P A K R I S V K D A L R H P Y F 299 GAAGATCTTAGCAGAGAAGATATAGCTAAATTTGAGCCTAATTAAGTTCATATGTACTGAATATGAAAA 966 E D L S R E D I A K F E P N Q V H M Y * 318 ACAAACAACAACTAAATTAATTAATTAACAAACAAATTAATTGCAAATAAATTTTTATTTAAGAATTGA 1035 TAATGAATGAATGAAA 1051 Fig. 3-1. Nucleotide sequence of TtCDKl, and the predicted amino acid sequence of the gene product, TtCdkl . The conserved 'PSTAIRE' region is double underlined. Putative phosphorylation sites, Thr21, Tyr22 and Thrl73 in TtCdkl , equivalent to Thrl4, Tyrl5 and Thrl61 of human CDK1, respectively, are indicated by bold italics. "*" indicates the stop codon TGA. The GenBank accession number of this sequence is AF157636. 99 Two introns were found within TtCDKl coding region by comparing its cDNA and genomic sequences. They all possess the consensus 573' splicing sites, GT/AG. Their sizes are 265 bp and 547 bp, respectively, much larger than those in P. tetraurelia genes (Russell et al., 1994; Tang et al., 1995; Zhang and Berger, 1999). The first intron is located in the same position as the second of two introns in PtCDKl. 3.2.2 TtCDKl is a member of the multi-gene family of CDKs in T: thermophila (a). There are at least two Cdk-relatedproteins in T. thermophila. Anti-PSTAIRE antibody detected two polypeptides of 35 kDa and 37 kDa, respectively, on a Western blot of T. thermophila total protein lysate (Fig. 3-2). The smaller peptide is much more abundant than the larger one. p35 is unlikely a proteolytic cleavage product of p37, considering their distinct pl3™ c / binding properties (see below). This result is in accord with previous results (Fujishima et al., 1992), and suggests the presence of at least two classes of Cdks in T. thermophila. A polyclonal antibody was generated against a carboxyl terminal peptide of TtCdkl in order to characterize functions of the TtCdkl protein. This specific anti-TtCdkl antibody recognized a peptide that comigrated with the larger p37 peptide recognized by the anti-PSTAIRE antibody (Fig. 3-2). This is in agreement with the predicted molecular mass for TtCdkl based on its primary sequence, suggesting TtCdkl is the large polypeptide recognized by anti-PSTAIRE antibody. This anti-Cdkl antibody recognizes only p37 and shows no cross-reaction with p3 5. 100 KDa ^ 47.7 — 34 .6 — Fig. 3-2. T. thermophila contains two anti-PSTAIRE reactive polypeptides: p37 and p35. Total cell lysate was resolved on SDS-PAGE, and transferred to Immobilon-P membrane. The membrane was probed with either anti-PSTAIRE antibody (anti-PSTAIRE lane) or anti-TtCdkl polyclonal antibody (anti-TtCdkl lane). K D a Anti-PSTAIRE W P i B 4 7 . 7 34 .6 Anti-TtCdkl Fig. 3-3. T. thermophila Cdks exhibit different affinities for yeast p i3 s protein. After incubating cell lysate with excess amount of pl3™c 7 agarose beads, both bound (P: precipitate) and unbound fractions (S: supernatant) along with total lysate (W: whole cell lysate) were loaded onto SDS-PAGE and then immunoblotted with either anti-PSTAIRE (Panel A) or anti-TtCdkl antibodies (Panel B). 101 (b). T. thermophila Cdks have different affinities for yeast pi3SUC protein. As shown in Fig. 3-3A, among the two peptides recognized by anti-PSTAIRE antibody, only p37 could be precipitated by yeast pl3™ e 7. p35 remained in supernatant after p37 was precipitated. These results indicate that p35 and p37 differ in their pl3sucl binding affinities. In order to test directly the affinity of TtCdkl for pl3' v" c y, I substituted the anti-PSTAIRE antibody used in the previous experiment with the anti-TtCdkl specific antibody. As shown in Fig. 3-3B, TtCdkl protein is retained on yeast pl3's"c/-Sepharose beads. This result indicates that TtCdkl is the p l3 S H e / binding Cdk in T thermophila, and confirms that TtCdkl corresponds to the p37 recognized by anti-PSTAIRE antibody. 3.2.3 Periodic expression of TtCdkl protein correlates with activation of its histone HI kinase during the vegetative cell cycle. The expression of Cdk proteins in cells of most of eukaryotes including yeast, vertebrates and P. tetraurelia is not regulated in a cell cycle stage-specific manner (Draetta et al, 1989; Tang et al, 1997; Zhang and Berger, 1999). Periodic activation of Cdk kinase activity is determined by post-translational modifications, such as cyclin binding, reversible phosphorylation and association with Cdk inhibitors (CKIs). With the anti-TtCdkl specific antibody, the protein level of TtCdkl was monitored in elutriation synchronized cell populations. Macronuclear D N A synthesis, as detected by the incorporation of [ H] thymidine into macronuclei, in the mass synchronized cell populations occurred normally in the middle of the cell cycle and took about 1 h at 30°C, suggesting no interference occurred during elutriation process on the cell cycle progression (Berger, unpublished data). The median time of cell division was about 3.0 h post elutriation (Fig. 3-4C). A total of 8 samples were collected at 30 min intervals, starting 30 min after elutriation. Protein lysates were prepared for each sample, 102 and analyzed by Western blotting with anti-TtCdkl antibody. To my surprise, the TtCdkl protein exhibited cell cycle-stage dependent expression during the vegetative cell cycle. As shown in Fig. 3-4A, TtCdkl was barely detectable early in the cell cycle. Its protein level began to increase gradually, starting from 2 h post elutriation, and reached a peak at ~3 h. At 3.5 hr when cells had entered a new cell cycle, TtCdkl protein level declined dramatically. This observation is in sharp contrast to the situations in most other eukaryotic cells including P. tetraurelia where the Cdk proteins are kept at approximately constant level over the vegetative cell cycle (Tang et al., 1997; Zhang and Berger, 1999). Consistent with this finding, a similar pattern of increase and decrease was also observed for the amount of TtCdkl protein retained by the yeast pl3™ e ; beads over the course of the cell cycle (data not shown). This result also implies that the pl3' s m ;-binding affinity of TtCdkl does not change appreciably over the course of the cell cycle. Moreover, I noticed the TtCdkl protein could be resolved into two closely-spaced bands between 2.5 to 3 hr post-elutriation when its level was high (Figs. 3-4A). The appearance of the doublet may represent alternatively phosphorylated forms of the TtCdkl protein, as seen for human CDK2 and CDK1 (Elledge et al, 1992), and imply that changes in the TtCdkl phosphorylation state occur during the cell cycle. However, no appreciable signal was detected in an attempt to examine phosphotyrosine in TtCdkl precipitated by p l 3 s u e 7 using the 4G10, the monoclonal antibody that has been used extensively for detecting tyrosine phosphorylation (Upstate Biotechnology, Lake Placid, NY). 103 1 2 3 4 5 6 7 8 AS B TtCdkl Histone H1 120 -r UM 100 -S 80 • 2 L L o 60 -LU CD < H 40 -2 111 RC 20 -U J C L 0 -1 1.5 2 2.5 3 3.5 TIME AFTER ELUTRIATION (h) -TtCdkl protein -TtCdkl kinase -Ar - Cell density Fig. 3-4. TtCdkl protein level and its histone HI kinase activity during the vegetative cell cycle. Aliquots of synchronized cells were taken at 0.5-h intervals, starting from 0.5 h post elutriation, to determined the cell density of the samples (C); protein level by western blot analysis using anti-TtCdkl antibody (A) and histone HI kinase activity present in TtCdkl immunoprecipitates (B). AS: TtCdkl protein level in asynchronized sample. Protein level and kinase activity were quantitated as described in Materials and Methods (C). (Note: this figure presents composite results derived from different batches of synchronized samples). 104 In accord with its kinase-like sequence, TtCdkl immunoprecipitate displayed phosphorylation activity towards bovine histone HI . No activity was observed when immunoprecipitation was performed with preirnmune serum (data not shown). To investigate in greater detail the dynamic changes of TtCdkl kinase activity during the cell cycle, I performed immunoprecipitation with anti-TtCdkl antibody in the lysates of elutriation synchronized samples. As shown in Fig. 3-4B, the TtCdkl kinase activity displayed cell cycle-stage dependent oscillation that is correlated with its protein level. The kinase activity, shown as the extent of phosphorylation of bovine histone HI , was barely detectable at the beginning of the cell cycle. An activity peak was observed at about 3 h post-elutriation when most cells had undergone cytokinesis. By judging from the quantitative data, TtCdkl kinase activity is closely correlated with its protein level (Fig. 3-4C). Whether and how post-translational modifications including cyclin binding and reversible phosphorylation contribute to TtCdkl kinase activation remains unknown. 3.2.4 TtCdkl protein is spatially associated with basal body domains in the cell cortex The TtCdkl specific antibody was used to visualize the localization of TtCdkl in vegetative T. thermophila cells by indirect immunofluorescence microscopy. Unlike most Cdks from other eukaryotic cells examined so far, TtCdkl was not detectable in micronuclei, macronuclei or cytoplasm under the resolution that could be achieved by the techniques used in this study (Fig. 3-5A, B). The anti-TtCdkl immunostaining signal appeared to be limited to more or less circular halo-like domains associated with the basal bodies of the ciliary rows of the cortex (Fig. 3-5A). The TtCdkl protein appeared to be localized to a region adjacent to, but not within basal bodies. The circular TtCdkl domains have a non-staining center, presumably corresponding to the basal body region. This circular TtCdkl staining zone was typically thinner 105 at the anterior edge and in some cases incomplete, giving a horseshoe-shaped appearance. In dividing cells smaller faintly staining dots were observed in positions at which newly formed basal bodies were expected, lying between basal bodies with fully developed TtCdkl staining domains. The TtCdkl protein was also associated with the polykineties (membranelles) and the haplokinety (undulating membrane) of the oral apparatus, an association that is clearest in oral structures of dividing cells (Fig. 3-5B, F). No staining was observed along the fission line in dividing cells where no basal bodies are located (Fig. 3-5B). No change was observed in the TtCdkl localization throughout the vegetative cell cycle. Double staining with anti-TtCdkl and anti-a-tubulin antibodies revealed a general correspondence between the sites of the basal bodies of the ciliary rows and the TtCdkl staining domains (Fig. 3-5D). Some basal bodies associated with the peripheral TtCdkl staining appeared yellowish due to overlap of the red (a-tubulin revealed by Texas Red-conjugated secondary antibody) and green (TtCdkl revealed by FITC-conjugated secondary antibody) fluorescing signals. These double-stained basal bodies all possess red-staining transverse microtubule bands extending to the cell's left, which are usually found in association with mature basal bodies. Other basal bodies, presumably those associated with the faintly staining dots seen in early dividers, were stained mostly red, due to little TtCdkl antigen associated with those basal bodies. These single-stained basal bodies, more abundant in the central and posterior regions of the cortex than anterior region, are usually located one or two positions anterior to double-stained basal bodies and generally do not possess transverse microtubule bands, although occasionally the posterior of two has a short one. I believe that the basal bodies without significant amounts of TtCdkl antigen associated with them are newly developed basal bodies. The weak signals around nascent basal bodies compared to the much stronger ones in the 106 domains surrounding mature basal bodies suggest that the deposition of TtCdkl occurs as basal bodies and associated cytoskeletal structures undergo their maturation. 3.2.5 Functional characterization of TtCDKl gene by targeted gene knockout in somatic macronuclei (a). Selection and characterization of the TtCDKl knockout cell lines Following bombardment, cells were resuspended in Neff growth medium and plated in 96-well microtiter plates. Paromomycin was added to final concentrations of 20 ug/ml, 50 rig/ml or 100 ug/ml 7 h after bombardment following incubation of cells at 30°C. While no paromomycin-resistant clones were observed in either 50 or 100 ug/ml paromomycin, more than a dozen drug-resistant clones were picked up in 20 ug/ml paromomycin 6-7 days after bombardment when all control cells that were bombarded with pBluescript vector sequence had died. I chose one drug-resistant cell line, T tCDKIKO, for further analysis. Southern blot analysis using a specific probe for the TtCDKl gene was performed with AWe/-digested genomic D N A isolated from T tCDKIKO grown in 50 ng/ml and 100 ug/ml paromomycin (Figs. 3-6; 3-7A, B). In wild-type cells, the expected single band of 1.7 kb was detected. In T tCDKIKO in either concentration of paromomycin, the intensity of the wild-type band was reduced and a larger band of 3.1 kb, resulting from neomycin-resistance cassette integrated into the TtCDKl locus, was present. The neomycin resistance-specific probe hybridized with only the larger band in each case, suggesting that neomycin-resistance cassette 107 Fig. 3-5. Localization of the TtCdkl protein in both wild-type and T t C D K l K O cells. Vegetative growing T. thermophila wild-type cells were stained with anti-TtCdkl (A and B) or preimmune serum (C), or double-stained with anti-TtCdkl antiserum (green) and anti-a tubulin monoclonal antibody 15D3 (red) (D). The TtCdkl protein is localized to circular domains associated with mature basal bodies of the ciliary rows and associated with the polykineties (membranelles) and the haplokinety (undulating membrane) of the oral apparatus. T t C D K I K O cells were stained with anti-TtCdkl antiserum (E and F), displaying specific phenotypes including bending of ciliary rows in the cell cortex, chromatin decondensation in both nuclei and decreasing intensity of TtCdkl stain. Nuclei in A, B, C, E and F were counter-stained with propidium iodide. Images were viewed and photographed under a confocal microscope. 108 1* » . * *> iiZ • • * **.--. ',. • * * * <* » «•. . - < 1 • \ ' v # * - • - •« '+ m m A B • c * » *>* _ V * > , *M*fcV n , - *• E F 109 was inserted only in the targeted gene. Comparing the intensities of the knockout and wild-type bands, more than 50% of macronuclear TtCDKl gene copies were disrupted in T tCDKIKO. Increasing the paromomycin concentration from 50 ug/ml to 100 ixg/ml resulted in further replacement of endogenous wild-type copies with the knockout construct (lane KO50 vs KO100). The observation that the endogenous TtCDKl alleles could not be completely replaced in T tCDKIKO even after prolonged selection in paromomycin suggests that the TtCDKl gene is essential for cell survival and, therefore, T tCDKIKO is a partial knockout transformant. Consistent with this, the wild-type alleles were fully restored in T t C D K I K O cells after prolonged growth without selection pressure (data not shown). Co-integration of the transforming fragment into the flanking sequences of the target locus has been observed in several cases, leaving the endogenous gene functional (Brown et dl., 1999; Gaertig et al, 1994, 1995; Kahn et al, 1993). Therefore, to verify that T tCDKIKO was a true transformant resulting from gene replacement, Southern blot analysis using the TtCDKl gene probe was carried out with Bstl-digested T tCDKIKO genomic D N A (Figs. 3-6; 3-7C). In addition to 3.0 kb and 0.8 kb wild-type bands, a 2.2 kb band expected from gene replacement appeared in T tCDKIKO, suggesting gene replacement, not co-integration, occurred in T tCDKIKO. Western blotting of protein lysate with anti-TtCdkl specific antibody showed -20% and -70% reduction in the expression of the T t C D K l gene in T t C D K I K O cells grown in 50 ug/ml and 100 ug/ml paromomycin, respectively, which is in accord with the partial knockout of the endogenous T t C D K l gene (Fig. 3-8). (b). Multiple deficiencies of the TtCDKIKO cells upon the reduction of TtCDKl expression 110 Since the TtCdkl protein was localized to the domains associated with the basal bodies of ciliary rows in the cortex, immunofluorescence microscopy using anti-TtCdkl antibody was employed to examine possible phenotypic effects upon partial TtCDKl knockout in the T tCDKIKO cells. As expected from reduced expression of the TtCDKl gene in T tCDKIKO based on western blot analysis data, the overall intensity of the TtCdkl protein staining was weaker in the T tCDKIKO cells than that in wild-type cells (Fig. 3-5E, F). Moreover, in contrast to the relatively straight alignment of basal bodies in wild-type cells, some degree of bending and buckling of ciliary rows was observed in most of the T t C D K I K O cells. No decrease in the number of basal bodies was noticed in the knockout cells, suggesting the TtCdkl protein might not be involved in the duplication of basal bodies. Moreover, a consistent correlation between the reduced TtCDKl expression and an increase of propidium iodide-stained areas in both types of nuclei was found in T tCDKIKO cells. Both macro- and micronuclear D N A contents of T tCDKIKO cells are almost indistinguishable from those of wild-type cells as determined by flow cytometry of propidium iodide-stained nuclei (Fig. 3-9). Thus, the increase in the propidium iodide-stained area in both nuclei in TCDK1KO cells is most likely due to the chromatin decondensation that accompanies the reduced expression of TtCDKl. No D N A fragmentation was detected on agarose gels (data not shown). This observation is reminiscent of the phenotypes of AH1 (macronuclear linker histone HHO gene knockout) and A M i c L H (micronuclear linker histone MLH gene knockout) cells, in which decondensation of either macro or micronuclear chromatin was detected (Shen et al, 1995). I l l S 3 5 1 9 oo T3 bo 3 oo PQ £ PQ Z PQ J I I I L 1.7 0.8 3.0 T3 oo neo 3.1 2.2 3.0 Wildtype PQ u PQ o W PQ PQ Z PQ I I I I L Knockout Fig. 3-6. Diagram of portions of the macronuclear wild-type TtCDKl gene (top) and the knockout construct (bottom). The sites of the enzymes used to digest genomic DNAs are indicated on the D N A sequences, and the fragments with expected sizes (kb) shown under the sequences. 112 WT KO kb 3 — . , WT KO50 KO100 WT KO50 KO100 kb 4 -1.6— 2 — • 1.6— % i A B 0.5— # C Fig. 3-7.Genomic Southern blot analysis to demonstrate partial disruption of the TtCDKl gene in T t C D K I K O cells. Genomic DNAs from wild-type cells (lane WT ) and a knockout cell line grown in 50 ug/ml (KO50) and 100 ug/ml paromomycin (KO100) digested with Ndel were separated on agarose gel and probed with the TtCDKl-specific probe (panel A) and neomycin-resistance gene probe (panel B), respectively. Genomic DNAs from wild-type (WT) and the knockout cells (KO) digested with BstBl were resolved on agarose gel and probed with the TtCDKl-specific probe to demonstrate the gene replacement event (panel C). WT KO50 KOI 00 TtCdkl Fig. 3-8. The TtCdkl protein level detected by western blot analysis in wild-type (WT) and T tCDKIKO cells grown in 50 ug/ml (KO50) and 100 ug/ml paromomycin (KO100). Total protein lysates were resolved on SDS-PAGE and probed with anti-TtCdkl antibody. 113 D N A Content Fig. 3-9. Macronuclear and micronuclear D N A contents of both wild-type (WT) and T t C D K l K O (KOI00) cells, as measured by flow cytometry. No apparent difference of their respective D N A content between wild-type cells (upper panel) and T t C D K I K O cells grown in 100 pg/ml paromomycin (lower panel) was observed in either macro- or micronuclei. 114 3.3 DISCUSSION Although Tetrahymena was the first eukaryotic cell to be successfully synchronized through serial heat shock treatment (T. pyriformis: Scherbaum and Zeuthen, 1954; T. thermophila: Holz et al., 1957), and T. pyriformis was used in many early cell cycle studies (Frankel et al., 1976; 1977), Tetrahymena was later replaced by more morphologically 'typical' eukaryotic cells such as yeast and mammalian cells, partially due to the complexity resulting from the nuclear dimorphism of ciliates. With the identification of Cdk/cyclin as conserved central regulators of cell cycle in these 'typical' eukaryotic cells, several groups have returned to this ciliate and identified both Cdk- and cyclin-like proteins in T. thermophila but without obtaining gene sequences of either class (Roth et al., 1991; Williams and Macey, 1991). The reason for exploring the molecular mechanisms of the T. thermophila cell cycle following the isolation and characterization both Cdks and cyclins in P. tetraurelia is not simply an issue of documenting species variation. T. thermophila offers a more convenient model system to work with in a sense of feasibility of examining in vivo functions of cloned genes, thanks to the recent development of targeted gene disruption techniques in T. thermophila (Cassidy-Hanley et al., 1997; Garetig and Gorovsky, 1992, Garetig et al., 1994; Tondravi and Yao, 1986). In this study, I have cloned a gene that encodes a Cdk-like protein from the T. thermophila genome using a homology based cloning approach. The striking overall homology of the TtCdkl protein with Cdk homologues from other eukaryotes, and in particular, the presence of the PSTAIRE region (GVPSTALREISILKE in TtCdkl), which is the hallmark of Cdks, leaves no doubt that TtCdkl is a Cdk homologue in T. thermophila. Variations in the PSTAIRE region were observed in the Cdk homologues from Dictyostelium discoideum 115 (Michaelis and Weeks, 1992), Plasmodium falciparum (Ross-MacDonald et ah, 1994), Entamoeba histolytica (Lohia and Samuelson, 1993), Trypanosoma brucei (Riley et al., 1993), and P. tetraurelia (Tang et al., 1995). Homologues of the typical regulatory phosphorylation sites, including Thrl4, Tyrl5 and Thrl61 in human CDK1, are located in corresponding positions within TtCdkl , suggesting the involvement of phosphorylation/dephosphorylation in regulating TtCdkl kinase activity. In agreement with this, the migration rate of TtCdkl protein on SDS-PAGE changed during the cell cycle, implicating possible changes in its overall phosphorylation state. The failure of the anti-phospho tyro sine antibody (4G10) to detect any phosphorylation on tyrosine residues on TtCdkl associated with pl3™c 7 could be due to phosphorylation on residues other than tyrosine, such as threonine(s). Almost all the Cdks identified so far need to bind to cyclin partners to become active. In P. tetraurelia, two classes of distinct mitotic cyclin homologues have recently been identified (Zhang et al., 1999; Chapter 2). They form complexes with different Cdk catalytic subunits, and activate kinase activity at different specific cell cycle stages. In the TtCdkl sequence, most of the residues and domains essential for cyclin binding are conserved, suggesting the possible interaction with a cyclin. In fact, a sequence has been identified in T. thermophila, displaying high homology with the 'cyclin box' in other eukaryotic cyclins (Zhang et al., 1999). However, the close correlation between the profile of the TtCdkl protein expression and that of its kinase activation during the vegetative cell cycle raises the question as to roles of post-translational modifications such as cyclin binding and phosphorylation in TtCdkl enzymatic activation. Roth et al. (1991) reported an anti-PSTAIRE reactive peptide (p36) that bound to yeast pl3' v" e ; and exhibited protein kinase activity towards both bovine histone HI and macronuclear 116 linker histone. Based on the pl3™c binding property, it seems likely that this p36 polypeptide corresponds to TtCdkl in this study. The absence of the smaller anti-PSTAIRE-reactive polypeptide (p35, non- pl3'™c; binding) in their study may be because their protein lysates were from macronuclei, not whole cells. Based on their results, it seems that TtCdkl should be, if not exclusively, localized in macronucleus. Contrary to my expectation, TtCdkl protein appears to be associated with cytoskeletal regions adjacent to basal bodies in the somatic cortex and is also found within the oral apparatus, but not in either nucleus, as revealed by indirect immunofluorescence microscopy with anti-TtCdkl antibody. However, the contradiction may be resolved if there are additional Cdks other than p35 and p37 present in T. thermophila. This is not completely unlikely, considering two Cdks (PtCdk2 and Cdk3) are present in P. tetraurelia with similar molecular weights but distinct pl3™c /-binding properties. The more-or-less circular halo-like domains of the TtCdkl protein associated with somatic basal bodies appear very similar to the K-antigen domains decorated by mAb 424A8 antibody (Williams et al. 1990). These domains were demonstrated by immunogold binding and electron microscopy to be localized within the membrane-skeletal layer (Williams et ah, 1990). The detailed similarity between the K-antigen and TtCdkl domains strongly suggests that the TtCdkl protein, like the K antigens, is localized to the specialized regions of the membrane skeleton (epiplasm) that surround the basal bodies of the ciliary rows. The similarity also extends to the development of these domains. Williams et al. (1990) found that the K-antigen domains associated with newly formed basal bodies are much smaller than those of more mature basal bodies. The results with both single staining with anti-TtCdkl antibody and double staining with anti-TtCdkl and anti-a-tubulin antibodies are very similar. The possession of transverse microtubule bands suggests the double-stained basal bodies are mature basal bodies 117 with a full complement of accessory structures, whereas the lack of full-length transverse microtubule bands and their localization relative to double-stained basal bodies suggests that the basal bodies staining only with anti-a-tubulin are newly formed. The probable association of TtCdkl protein with the epiplasmic domains that surround mature basal bodies suggests that TtCdkl may be involved in the maturation of the complex membrane-skeletal layer within which all cytoskeletal structures are embedded (Williams et al., 1990; Frankel, 1999). It may also play a role in the development of the oral apparatus. Consistent with these suggestions, the fairly broad peak of TtCdkl histone HI kinase activity associated with timing of division-related cortical morphogenesis encompasses the period of formation of the oral primordium and the maturation of many new somatic ciliary units (Frankel, 1981; Williams et al., 1990). The role of protein kinases in the establishment of morphogenetic patterns in Tetrahymena or other organisms is not well understood. However, the association of Cdk/cyclin complexes with centrosomes and the apparent requirement for CDK2/cyclin E activity for centrosome duplication in animal cells (Bailly et al., 1989; 1992; Hinchcliffe et al., 1999) indicates that the association of a Cdk with basal body domains in Tetrahymena may represent a common rather than a unique phenomenon. In the ciliate case, TtCdkl might play a role in the differentiation of the complex membrane-skeletal layer that may be involved in cortical morphogenesis in this organism (Frankel, 1999). The buckling of ciliary rows observed in the T t C D K l partial knockout cells may reflect deficiencies in the formation of this layer. Recent studies of the distribution of a serine/threonine kinase activity in the cortex of T. thermophila, and the effects of inhibitors of protein kinases on division associated morphogenesis suggest that in Tetrahymena, as in other organisms, protein kinases play a specific and important role in establishing the morphogenetic pattern (Kaczanowska et al. 118 1999). Phosphorylation of basal body associated structures located in the cortex and the oral apparatus has been observed throughout the vegetative cell cycle in both T. thermophila and P. tetraurelia, as demonstrated by staining cells with MPM-2 antibody, an antibody which recognizes phosphorylated epitopes in microtubule organizing centers (MTOCs) in variety of organisms (Huelsman et al., 1992; Keryer et al., 1987). Direct evidence has been gathered for cell cycle-dependent phosphorylation of the structural proteins in the cortex including the ciliary rootlets (Sperling et al., 1991). However, the nature of the protein kinase(s) responsible for the phosphorylation events and their roles in ciliate morphogenesis remains an issue of speculation. My demonstration of the probable association of TtCdkl with the membrane-skeletal domains surrounding the basal bodies in the cell cortex and the buckling of ciliary rows upon reduced expression of the TtCDKl gene provides the first evidence for the involvement of a Cdk kinase in the cortical morphogenesis of ciliates prior to cell division. It has been demonstrated that both pl3™ c /-bound p36 and TtCdkl exhibit protein kinase activity towards the macronuclear histone HI protein (Roth et al., 1991; Mizzen, personal communication). Disruption of the macronuclear copies of the histone HI genes leads to decondensation of the macronuclear chromosomes. It is believed that phosphorylation of the macronuclear histone HI is essential for chromosome condensation/decondensation. The decondensation of both macro and micronuclear chromosomes observed in the T tCDKIKO cells suggests an indirect role of TtCdkl in chromatin condensation, possibly through phosphorylation of linker histones in both nuclei. The localization of the TtCdkl protein in epiplasmic domains associated with the basal bodies in the cell cortex throughout the cell cycle ehminates the possibility of direct involvement of TtCdkl in this process. However, a protein kinase activated by TtCdkl could be directly responsible for linker histone phosphorylation. 119 DISCUSSION AND FUTURE PROSPECTS A. GENERAL DISCUSSION The ciliate cell cycle typically consists of an initial period of growth, followed by a period of growth and morphogenesis (macronuclear and micronuclear D N A synthesis, stomatogenesis and somatic cortex morphogenesis) and ending with cell division (macronuclear amitosis, micronuclear mitosis, stomatogenesis and cytokinesis) (Adl and Berger, 1996). In contrast to the situation in higher eukaryotic cells where various cell cycle events are tightly coupled with each other through various checkpoint controls, all these processes occur independently within the ciliate cell and can be unlinked both with inhibitors and in mutants. Of particular interest is the question how the ciliate coordinates the cell cycle events occurring in different compartments of the single cell. While the jury is still out on this broad question, the supposition that the basic molecular workings of the cell division cycle are similar in all eukaryotic cells has provided a convenient starting point for characterizing the molecular nature of cell cycle control in ciliates. As the master regulators of the eukaryotic cell cycle, Cdks have been demonstrated to play vital roles in controlling and coordinating various cell cycle events, ensuring that cells progress through sequential stages of the division cycle in an orderly fashion (Stern and Nurse, 1996). The activity and specificity of Cdks are controlled at many levels, making them versatile regulators that are capable of integrating diverse cellular and extracellular signals. This makes Cdks excellent candidates for mediating control of the cell cycle in ciliates. Therefore, as a first step to elucidate the molecular basis of the ciliate cell cycle regulation, the Berger's lab has focused on demonstrating the presence of Cdks and cyclins and characterizing their roles in the ciliate cell cycle. 120 By taking advantage of high degree of conservation of both Cdks and cyclins in their amino acid sequences, I utilized homology-based cloning approaches including degenerate PCR, anchor PCR and library screening, to identify a novel Cdk gene and two classes of mitotic cyclin genes from ciliate P. tetraurelia in this study. Combined with previous results (Tang et al., 1994; 1997), at least three classes of Cdks and two classes of mitotic cyclins have been demonstrated in P. tetraurelia. This is the first time that cyclins have been cloned in ciliates, and is also the first case, to my best knowledge, that multiple Cdks have been shown to engage in cell cycle control in unicellular lower eukaryotes. In order to characterize the functions of the ciliate Cdk(s) in vivo, another holotrichous ciliate T. thermophila was introduced into the study, due to availability of highly efficient D N A transformation techniques in this particular ciliate. Following the isolation of a Cdk-like sequence, TtCDKl, in T thermophila, a partial knockout cell line was constructed using the somatic biolistic transformation technique. Detailed analysis of this knockout cell line revealed the possible involvement of the TtCdkl protein in the maturation of the membrane-skeletal domains associated with basal bodies of the ciliary rows in the cortex and in D N A condensation in both types of nuclei. Considering the divergence between ciliates and typical eukaryotic cells in both evolutionary history and cell cycle organization, my results not only extend the omnipresence of Cdk/cyclins to this unique, diverse, unicellular group and confirm that evolution of Cdk/cyclins occurred before the separation of ciliates from the lineage that then gave rise to higher eukaryotes, but also reveals some interesting roles that ciliate Cdk(s) play in the cell cycle and lays the foundations for further investigation of the molecular basis of the unusual cell cycle control system in ciliates. 121 1. Multiple Cdk/cyclin complexes involved in the unicellular cell cycles of ciliates. In the yeasts, S. pombe and S. cerevisiae, a single Cdk controls different cell cycle events through sequential association with stage-specific cyclin subunits. The identification of multiple Cdks in the ciliates, P. tetraurelia and T. thermophila, along with two different classes of mitotic cyclin homologues in P. tetraurelia poses the question as to why this unicellular organism requires multiple Cdks and cyclins for the control of the cell cycle. The unique cellular structure of ciliates, with different temporally and spatially regulated cell cycle events in macronucleus, micronucleus, cytoplasm and cell cortex, seems to partly justify my findings. The coincidence of the timing of the kinase activities associated with PtCdkl, PtCdk2 and PtCdk3 with IDS, cell division, and PCD, respectively, clearly supports this notion. Moreover, it seems more likely that multiple Cdks are the norm, and that yeasts with only one Cdk are the exception. Coordination of the Cdk kinase activities must be essential for the integration of various cell cycle events occurring in different compartments within the ciliate cell at cell division. Until now, little is known about the control of cell cycle events in the micronucleus. The cell cycle events such as D N A replication and mitosis occurring in micronuclei seem to be typical of higher eukaryotic cells. Surprisingly, studies on the linker histones in T. thermophila have shown that macronuclear histone HI but not micronuclear linker histone contains sequences resembling Cdk consensus phosphorylation sites whereas each of the micronuclear linker histone proteins (a, p\y, and 8) contain only typical cAMP-dependent protein kinase A (PKA) phosphorylation sites in their carboxyl termini (Wu et al., 1994). This leads to the tentative conclusion that 122 the micronuclear cell cycle events are controlled by P K A instead of Cdk(s). In agreement with this conclusion, no Cdk activity was found in either P. tetraurelia or T. thermophila in association with micronuclear cell cycle events. However, this point still requires further examination, given the unusual structural properties of the micronuclear linker histones in T. thermophila (Allis et al., 1979; 1984). 2. Coordination of the cell cycle events occurring on the cell cortex and within nuclei through a cyclin-dependent protein kinase. The striking similarity of the ciliate Cdks with their counterparts in higher eukaryotes in terms of sequence and biochemical properties leaves no doubt that they belong to the Cdk family. Furthermore, the correlation between their associated protein kinase activities with IDS, PCD or cell division in P. tetraurelia clearly suggests their involvement in ciliate cell cycle regulation. However, the question remains open as to what, exactly, these Cdks do in the ciliate cell cycle. To address this question, T. thermophila was introduced into the study. With the recent development of targeted gene disruption techniques, T. thermophila has become a more favorable organism as opposed to P. tetraurelia for characterizing gene function, due to its unique characteristic of phenotypic assortment (reviewed in Sonneborn, 1974). One of the most striking results in the study of the TtCdkl protein of T. thermophila was its association with the membrane skeletal domains of the basal bodies in ciliary rows and the polykineties and the haplokinety of the oral apparatus. No detectable protein was observed in macronucleus, micronucleus or cytoplasm in this study. Similar observations were made for PtCdk2 (Zhang and Berger, unpublished data). The association of Cdk/cyclin complexes with centrosomes and the apparent requirement 123 for CDK2/cyclin E activity for centrosome duplication in animal cells (Bailly et al., 1989; 1992; Hinchcliffe et al, 1999) indicates that the association of a Cdk with basal body domains in Tetrahymena may represent a common rather than a unique phenomenon. Preferential binding of the TtCdkl protein to the epiplasmic domains associated with mature basal bodies in the cell cortex, as revealed by double labeling of TtCdkl and a-tubulin, suggests the possible involvement of the TtCdkl in the maturation of the complex membrane-skeletal layer within which all cytoskeletal structures are embedded. To examine this hypothesis, I constructed a T. thermophila cell line, T t C D K I K O , in which more than 50% of endogenous TtCDKl gene copies in the macronucleus were replaced by a neomycin knockout construct. Complete replacement of wild-type alleles could not be achieved even after prolonged selection in paromomycin, suggesting that TtCDKl is an essential gene. Consistent with the proposed role of TtCdkl in the maturation of the membrane-skeletal layer, some degree of bending and buckling of ciliary rows was observed in the TtCDKl knockout cells, which may be due to deficiencies in the formation of this layer. Moreover, the defects in the basal body alignment within ciliary rows became more severe as paromomycin concentration increased. Detailed study of the basal bodies in T t C D K I K O cells by electron microscopy is needed to clarify this point. In spite of the absence of any detectable amount of the TtCdkl protein in either macro- or micronucleus, reduced expression of TtCdkl leads to the increase of propidium iodide-stained areas in both nuclei without the increase in D N A content, suggesting that TtCdkl may have a role in chromatin condensation in vivo. Cdk-mediated histone 124 phosphorylation has long been believed as a trigger to chromatin condensation (Bradbury et al, 191'4; Inglis et al, 1976; Matsumoto et al, 1980). The absence of the TtCdkl protein in both nuclei suggests that the involvement of TtCdkl in chromatin condensation must be indirect in T. thermophila. Previous studies have shown that macronuclear histone HI can be phosphorylated by the pl3™ c 7-binding Cdk (TtCdkl) in vitro whereas micronuclear linker histone appears to be phosphorylated by cAMP-dependent protein kinase A (PKA), as only P K A but not Cdk putative phosphorylation sites was found in its sequence (Roth et al, 1991; Sweet and Allis, 1993; Wu et al, 1994). The discrepancy between localization of the TtCdkl protein and nuclear chromosome phenotypes in T tCDKIKO cells could be reconciled if there were two separate pathways (possibly protein kinase cascades) mediating TtCdkl activity towards nuclei, with TtCdkl as the common component shared by the two pathways. In the macronucleus, TtCdkl might activate an unknown Cdk or pro line-directed protein kinase through unknown mechanism which in turn causes D N A condensation. On the other hand, TtCdkl might be responsible for P K A activation by an unknown mechanism and this in turn results in chromatin condensation in micronucleus. However, this hypothesis needs to be further tested by examining phosphorylation changes of linker histones of both macro- and micronuclei in T tCDKIKO cells. The involvement of a single TtCdkl in both maturation of basal-body domains in the cell cortex and chromatin condensation in nuclei suggests that it might play a role in the coordination of cell cycle events occurring in different compartments of the ciliate cell, which is very important for ciliate cell cycle controls. 3. Variations in the regulatory mechanisms for ciliate Cdks. 125 While the universality of the Cdk/cyclin motif has been demonstrated in ciliates, and the general picture of their involvement of the ciliate cell cycle control looks similar to that in other eukaryotic cells, the mechanisms required to regulate the ciliate Cdks appear to vary from enzyme to enzyme and in some cases seem to differ from those of typical Cdks. While the domains responsible for cyclin binding are conserved in PtCdkl sequence, no cyclin has been found so far in association with PtCdkl. Even though it is possible that the homology-based cloning approach failed to detect a less conserved and distinct class of cyclin complexed with PtCdkl (e.g. a G l cyclin), glycerol density gradient centrifugation data suggested that PtCdkl might exist as a monomer that exhibits kinase activity towards bovine histone HI (Tang et al., 1997). The TtCdkl protein exhibits higher sequence homology to PtCdk2, than to PtCdkl. In addition, both TtCdkl and PtCdk2 display maximal histone HI kinase activities late in the cell cycle. However, the TtCdkl protein exhibits affinity for the yeast pl3™ c / protein, while the PtCdk2 protein does not. More strikingly, unlike PtCdkl and PtCdk2, and most of other Cdks identified in other eukaryotes, the TtCdkl protein abundance is regulated in a cell cycle-dependent manner. Furthermore, the close correlation between protein level and kinase activity of TtCdkl raises the question as to whether the 'universal' post-translational modifications typically required for Cdk activation are necessary for TtCdkl activity. Similar observations have only been made on one other cell cycle-related protein kinase, the Aspergillus NimA (never-in-mitosis in Aspergillus nidulans), which encodes a 79 kDa protein kinase without significant sequence homology with Cdks. NimA functions as a positive regulator of CDK1 and 126 mitosis in that particular fungus (Ye and Osmani, 1997). A NimA homologue has been identified recently from T. thermophila (Wang et al., 1998). Despite the high degree of sequence identity between ciliate Cdks/cyclins and their counterparts in other eukaryotic cells, the mechanisms that regulate Cdk are not well conserved, suggesting that the regulatory mechanisms are a more recent evolutionary event than the acquisition of the gene itself. The issue here is that there are potentially lots of ways to regulate enzymatic activity. While higher organisms have settled on a precise and limited set of regulatory mechanisms, it is conceivable that in the very wide spectrum of lower eukaryotes there is range of regulatory processes active in regulation of their cell cycles. My work has supplied some intriguing 'snap shots' of regulation of Cdks in lower eukaryotic cells. Unfortunately, we do not yet have a good view of the whole range of regulatory processes. B . CONCLUSIONS Based on the observations I have made in this study, some important conclusions can be drawn: 1. In spite of the unusual cellular structure and cell cycle organization, the central components including Cdks and cyclins of the conserved cell cycle regulatory machinery across eukaryotic cells examined so far have been identified in two holotrichous ciliates, Paramecium tetraurelia and Tetrahymena thermophila. 2. Like their counterparts in higher eukaryotic cells, all the Cdks identified so far in ciliates exhibit histone HI protein kinase activity in vitro and each has a distinct activity profile, coinciding temporally with the major cell cycle events occurring over the course of the vegetative cell cycle. Likewise, the two mitotic cyclin homologues 127 isolated from P. tetraurelia display the classical cell cycle-dependent oscillations in their protein levels and each was found to be associated with distinct Cdk partner and resulted in the activation of kinase activity. 3. While the universality of Cdk/cyclin motifs in eukaryotic cell cycle control has been extended to ciliates by this study, the detailed mechanisms of regulating the ciliate Cdk kinase activities can vary somewhat from Cdk to Cdk, and differ in some respects from those in higher eukaryotic cells. This observation is consistent with the evolutionary divergence between ciliates and higher eukaryotes and within the ciliate phylum itself. 4. Phenotypic analysis of a TtCDKl partial knockout cell line has provided initial insights into the roles of a ciliate Cdk in vivo. The possible involvement of TtCdkl in the cortical morphogenesis prior to cell division and in chromatin condensation in both nuclei has been demonstrated through the analysis of a T. thermophila CDK1 knockout cell line by immunofluorescence microscopy. The fact that the two cell cycle events occurred in different compartments within a cell are regulated by a single Cdk suggests a possible mechanism employed by the ciliate cell to coordinate different cell cycle events within the ciliate cell. C. FUTURE PROSPECTS As a further step towards a more complete understanding of the molecular nature of the ciliate cell cycle, it is necessary to identify all the 'players', namely Cdks and cyclins, which are involved in its control. So far the PtCDK3 gene has not been cloned. Since the PtCdk3 kinase activity is associated with the PCD (Tang et al., 1994), the major control point of the P. tetraurelia cell cycle, identification of this Cdk could give us more 128 insights into the molecular basis underlying the commitment to cell division in P. tetraurelia. While homology-based cloning could still be a method of choice for isolating the PtCDK3 gene, the more direct approach would be to purify the protein by virtue of its unique pl3sucl binding property, and then sequence it to obtain the related D N A sequence information necessary for PtCDK3-specific PCR amplification. Similarly in T. thermophila, the isolation of additional Cdk genes including the one encoding p35 from its Cdk family and any further cyclin-like gene(s) would give a more complete picture about the molecular basis of cell cycle control in this organism. So far a partial cyclin-like sequence has been obtained in T. thermophila (see Chapter 2). The functional characterization of each Cdk and cyclin that has been and will be isolated from ciliates will continue to be the main story of ciliate cell cycle studies. Considering the recent emergence of several D N A transformation techniques in P. tetraurelia including homology-dependent gene silencing (Ruiz et al., 1998a, b) and antisense oligodeoxynucleotide (ODN) injection (Fraga et al., 1998; Hinrichsen et al., 1992), it should not take long before Paramecium researchers can find a way to routinely characterize essential genes in vivo in P. tetraurelia. Functional analysis of the Paramecium Cdks and cyclins coupled with characterization of additional Cdks and cyclins from T. thermophila may help us to explain the difference of cell cycle control in this two ciliates. One of the ultimate goals of the ciliate cell cycle studies is to investigate how cell cycle events occurring in different compartments of the ciliate cell are coordinated with one another, and the construction of double or even triple knockout cell lines should make it possible to dissect the interrelationship between various Cdks. The highly 129 sophisticated gene knockout techniques developed in T. thermophila have made this goal technically achievable. Therefore, identification and isolation of the whole cast of Cdk family members in T. thermophila should be an immediate goal in any follow-up studies. 130 BIBLIOGRAPHY 1. Adl, M . S. & Berger, J. D. 1992. Timing of micronuclear mitosis and its relation to commitment to division in Paramecium tetraurelia. Dev. Genet., 13:229—234. 2. Adl, M . S. & Berger, J. D. 1995. Comparison of methods of cell cycle analysis in Paramecium tetraurelia. J. Euk. Microbiol., 42:213—218. 3. Adl, S. M . & Berger, J. D. 1996. Commitment to division in ciliate cell cycles. J. Eukaryotic Microbiol., 43:11—86. 4. Adlakha, R. C , Wright, D. A. , Saharsrabuddhe, C. G., Davis, F. M . , Prashad, N . , Bigo, H . & Rao, P. 1985. Partial purification and characterization of mitotic factors from Hela cells. Exp. Cell Res., 160: 471—482. 5. Allen, R. D. 1969. The morphogenesis of basal bodies and accessory structures of the cortex of the ciliated protozoan Tetrahymena pyriformis. J. Cell Biol., 40:716—733. 6. Allen, S. L . , Altschuler, M . I., Bruns, P. J., Cohen, J., Doerder, F. P., Gaertig, J., Gorovsky, M . , Orias, E. & Turkewitz, A. 1998. Proposed genetic nomenclature rules for Tetrahymena thermophila, Paramecium primaurelia and Paramecium tetraurelia. Genetics, 149:459-462. 7. Allis, C. D., Allen, R. L . , Wiggins, J. C , Chicoine, L . C. & Richman, R. 1984. Proteolytic processing of Hl-like histones in chromatin: physiologically and developmentally regulated event in Tetrahymena micronuclei. J. Cell Biol., 99:1669— 1677. 8. Allis, C. D., Glover, C. V . C. & Gorovsky, M . A. 1979. Micronuclei of Tetrahymena contain two types of histone H3. Proc. Natl. Acad. Sci. USA., 76:4857—4861. 131 9. An, S. F., Franklin, D. & Fleming, K. A. 1992. Generation of dioxigenin-labelled double-stranded and single-stranded probes using the polymerase chain reaction. Mol. Cell. Probes, 6:193-200. 10. Andersen, H. A. & Zeuthen, E. 1971. D N A replication sequence in Tetrahymena is not repeated from generation to generation. Exp. Cell Res., 68:309—314. 11. Andersen, H . A. 1977. Replication and functions of macronuclear D N A in synchronously growing populations of Tetrahymena pyriformis. Carsberg Res. Commun., 42:225-248. 12. Arellano, M . & Moreno, S. 1997. Regulation of CDK/cyclin complexes during the cell cycle. Int. J. Biochem. Cell Biol, 29:559-573. 13. Arion, D., Meijer, L . , Brizuela, L . & Beach, D. 1988. cdc2 is a component of the M phase-specific histone HI kinase: evidence for identity with MPF. Cell, 55:371—378. 14. Bai, C , Richman, R. & Elledge, S. J. 1994. Human cyclin F. EMBO J., 13:6087— 6098. 15. Bailly, E. , Doree, M . , Nurse, P. & Bornens, M . 1989. p34 c d c 2 is located in both nucleus and cytoplasm; part is centrosomally associated at G2/M and enters vesicles at anaphase. EMBO J., 8:3985—3995. 16. Bailly, E., Pines, J., Hunter, T. & Bornens, M . 1992. Cytoplasmic accumulation of cychn B I in human cells: association with a detergent-resistant compartment and with the centrosome. J. Cell Sci., 101:529—545. 17. Baldin, V. , Lukas, J., Marcote, M . J., Pagano, M . & Draetta, G. 1993. Cychn D l is a nuclear protein required for cell cycle progression in G l . Genes Dev., 7:812-821. 132 18. Bates, S., Rowan, S. & Vousden, K. H. 1996. Characterization of human cychn G l and 02: D N A damage inducible genes. Oncogene, 13:1103—1109. 19. Beach, D., Durkacz, B. & Nurse, P. 1982. Functionally homologous cell cycle control genes in budding and fission yeast. Nature, 300:706—709. 20. Berger, J. D. & Kimball, R. F. 1964. Specific incorporation of precursors into D N A by feeding labeled bacteria to Paramecium aurelia. J. Protozool., 11:534—537. 21. Berger, J. D. & Rahemtullah, S. 1990. Commitment to autogamy in Paramecium blocks mating reactivity: implications for regulation of the sexual pathway and the breeding system. Exp Cell Res., 187:126—133. 22. Berger, J. D. 1971. Kinetics of incorporation of D N A precursors from ingested bacteria into macronuclear D N A of Paramecium aurelia. J. Protozool, 18:419—429. 23. Berger, J. D. 1979. Regulation of macronuclear D N A content in Paramecium tetraurelia. J. Protozool, 26:18—28. 24. Berger, J. D. 1988. The cell cycle and regulation of cell mass and macronuclear D N A content. In: Gortz, F£. D. (ed.), Paramecium. Springer-Verlag, Berlin. Pp. 97—119. 25. Berger, J. D. 1989. The cell cycle in lower eukaryotes. Curr. Opin. Cell Biol, 1:256— 262. 26. Bradbury, E. M . , Inglis, R. J., Matthews, H . R. & Langan, T. A . 1974. Molecular basis of control of mitosis cell division in eukaryotes. Nature, 249:553—555. 27. Bradford, M . M . 1976. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72:248-254. 133 28. Brizuela, L. , Draetta, G. & Beach, D. 1987. pl3™ c / acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. EMBO J., 6:3507-3514. 29. Broek, D., Bartlett, R., Crawford, K. & Nurse, P. 1991. Involvement of p34cdc2 in establishing the dependency of S phase on mitosis. Nature, 349:388-393. 30. Brown, J. M . , Marsala, C , Kosoy, R. & Gaertig, J. 1999. Kinesin-II is perferentially targeted to assembling cilia and is required for ciliogenesis and normal cytokinesis in Tetrahymena. Mol. Biol. Cell, 10:3081—3096. 31. Buck, V . , Russell, P. & Millar, J. B. 1995. Identification of a cdk-activating kinase in fission yeast. EMBO J., 14:6173-6183. 32. Cardoso, M . C , Leonhardt, H . & Nadal-Ginard, B. 1993. Reversal of terminal differentiation and control of D N A replication: cyclin A and Cdk2 specifically localize at subnuclear sites of D N A replication. Cell, 74:979-992. 33. Caron, F. & Meyer, E. 1985. Does Paramecium primaurelia use a different genetic code in its macronucleus? Nature, 314:185—188. 34. Cassidy-Hanley, D., Bowen, J., Lee, J. H. , Cole, E., VerPlank, L . A. , Gaertig, J., Gorovsky, M . A. & Bruns, P. J. 1997. Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics, 146:135—147. 35. Cavalier-Smith. 1993. Kingdom Protozoa and its 18 phyla. Microbiol. Rev. 57:953— 993. 36. Chou, Y.-Ff., Bischoff, J. R., Beach, D. & Goldman, R. D. 1990. Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin. Cell, 62:1063-1071. 134 37. Cleffmann, G. 1968. Regulierung der DNS-Menge im Makronukleus von Tetrahymena. Exp. Cell Res., 50:193—207. 38. Cleffmann, G. 1975. Amount of D N A produced during extra S phases in Tetrahymena. Chromosoma, 78:313-325. 39. Colasanti, J., Tyers, M . & Sundaresan, V . 1991. Isolation and characterization of cDNA clones encoding a functional p34 c d c 2 homologue from Zea mays. Proc. Natl. Acad. Sci. USA, 88:3377-3381. 40. Coleman, T. R. & Dunphy, W. G. 1994. Cdc2 regulatory factors. Curr Opin Cell Biol, 6:877-882. 41. Coleman, T. R., Carpenter, P. B. & Dunphy, W. G. 1996. The Xenopus Cdc6 protein is essential for the initiation of a single round of D N A replication in cell-free extracts. Cell, 87:53-63. 42. Connel-Crowley, L. , Solomon, M . J., Wei, N . & Harper, J. W. 1993. Phosphorylation independent activation of human cyclin-dependent kinase 2 by cychn A in vitro. Mol. Biol. Cell, 4:79-92. 43. D'Urso, G., Grallert, B. & Nurse, P. 1995. D N A polymerase alpha, a component of the replication initiation complex, is essential for the checkpoint coupling S phase to mitosis in fission yeast. J Cell Sci., 108 (Pt 9):3109-3118. 44. Dahmann, C. & Futcher, B. 1995. Specialization of B-type cyclins for mitosis or meiosis in S. cerevisiae. Genetics, 140:957—963. 45. Damagnez, V. , Makela, T. P. & Cottarel, G. 1995. Schizosaccharomyces pombe Mopl-Mcs2 is related to mammalian C A K . EMBO J., 14:6164-6172. 135 46. De Bondt, H . L. , Rosenblatt, J., Jancarik, J., Jones, H . D., Morgan, D. O. & Kim, S.-H . 1993. Crystal structure of cyclin-dependent kinase 2. Nature, 363:595—602. 47. Deshaies, R. J. 1995. The self-destructive personality of a cell cycle in transition. Curr Opin Cell Biol, 7:781-789. 48. Devault, A. , Martinez, A. M . , Fesquet, D., Labbe, J. C , Morin, N . , Tassan, J. P., Nigg, E. A. , Cavadore, J. C. & Doree, M . 1995. MAT1 ('menage a trois') a new RING finger protein subunit stabilizing cyclin H-cdk7 complexes in starfish and Xenopus C A K . EMBO J., 14:5027-5036. 49. Dirick, L . , Bohm, T. & Nasmyth, K. 1995. Roles and regulation of Cln-Cdc28 kinases at the start of the cell cycle of Saccharomyces cerevisiae. EMBO J., 14:4803—4813. 50. Doerder, F. P. & DeBault, L . E. 1978. Life cycle variation and regulation of macronuclear D N A content in Tetrahymena thermophila. Chromosoma, 69: 1-19. 51. Doerder, F. P., Frankel, J., Jenkins, L . M . & DeBault, L . E. 1975. Form and pattern in ciliated protozoa: analysis of a genie mutant with altered cell shape in Tetrahymena pyriformis, syngen 1. J. Exp. Zool, 192: 237—258. 52. Donovan, J. D., Toyn, J. H. , Johnson, A. L . & Johnston, L . H . 1994. P40 S D B 2 5 , a putative C D K inhibitor, has a role in the M / G l transition in Saccharomyces cerevisiae. Genes Dev., 8:1640-1653. 53. Draetta, G. & Beach, D. 1989. The mammalian cdc2 protein kinase: mechanisms of regulation during the cell cycle. J Cell Sci Suppl, 12:21—27. 54. Ducommun, B., Brambilla, P. & Draetta, G. 1991. Mutations at sites involved in Sucl binding inactivate Cdc2. Mol. Cell Biol, 11:6177-6184. 136 55. Dunphy, W. G. & Newport, J. W. 1988. Mitosis-inducing factors are present in a latent form during interphase in the Xenopus embryo. J Cell Biol, 106:2047—2056. 56. Dunphy, W. G. & Newport, J. W. 1989. Fission yeast pl3 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase. Cell, 58:181-191. 57. Dunphy, W. G., Brizuela, L. , Beach, D. & Newport, J. 1988. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell, 54:423—431. 58. Dutta, A. & Stillman, B. 1992. Cdc family kinases phosphorylate a human cell D N A replication factor, RPA, and activate D N A replication. EMBO J., 11:2189-2199. 59. Edwards, M . C., Wong, C. & Elledge, S. J. 1998. Human cyclin K, a novel RNApolymerase II-associated cyclin possessing both carboxy-terminal domain kinase and Cdk-activating kinase activity. Mol. Cell. Biol. 18:4291—4300. 60. El-Deiry, W. S., Tokino, T., Velculescu, V . E., Levy, D. B., Parsons, R., Trent, J. M . , Lin, D., Mercer, W. E., Kinzler, K. W. & Vogelstein, B. 1993. WAF1, a potential mediator of p53 tumor suppression. Cell, 75:817—825. 61. Elledge, S. J. & Spottswood, M . R. 1991. A new human p34 protein kinase, CDK2, identified by complementation of a cdc28 mutation in Saccharomyces cerevisiae, is a homolog of Xenopus E g l . EMBO J., 10:2653-2659. 62. Elledge, S. J., Richman, R., Hall, F. L. , Williams, R. T., Lodgson, N.& Harper, J.W. 1992. CDK2 encodes a 33-kDa cyclin A-associated protein kinase and is expressed before CDC2 in the cell cycle. Proc Natl Acad Sci U S A., 89:2907—2911. 63. Engler-Blum, G., Meier, M . , Frank, J. & Muller, G. A. 1993. Reduction of background problems in nonradioactive Northern and Southern blot analysis enables higher sensitivity than P32-based hybridizations. Anal. Biochem., 210:235—244. 137 64. Enoch, T. & Nurse, P. 1991. Coupling M phase and S phase: controls maintaining the dependence of mitosis on chromosome replication. Cell, 65:921—923. 65. Evans, T., Rosenthal, E. T., Youngblom, J., Distel, D. & Hunt, T. 1983. Cychn: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell, 33:389—396. 66. Ewen, M . E., Sluss, H . K., Sherr, C. J., Matsushime, H. , Kato, J. & Livingston, D. M . 1993. Functional interactions of the retinoblastoma protein with mammalian D-type cyclins. Cell, 73:487-497. 67. Fang, F. & Newport, J. W. 1991. Evidence that the Gl -S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. Cell, 66:731—742. 68. Feaver, W. J., Svejstrup, J. Q., Henry, N . L . & Kornberg, R. D. 1994. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK. Cell, 79:1103-1109. 69. Fesquet, D., Labbe, J. C , Derancourt, J., Capony, J. P., Galas, S., Girard, F., Lorca, T., Shuttleworth, J. Doree, M . & Cavadore, J. C. 1993. The M O 15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thrl61 and its homologues. EMBO J., 12:3111-3121. 70. Fisher, D. & Nurse, P. 1995. Cyclins of the fission yeast Schizosaccharomyces pombe. Semin. Cell Biol, 6:73—78. 71. Fisher, D. & Nurse, P. 1996. A single fission yeast mitotic cychn B p34 c < f c 2 kinase promotes both S-phase and mitosis in the absence of G l cyclins. EMBO J., 15:850— 860. 138 72. Fisher, R. P. & Morgan, D. O. 1994. A novel cychn associates with M015/CDK7 to form the CDK-activating kinase. Cell, 78:713-724. 73. Fisher, R. P. 1997. CDKs and cyclins in transition(s). Curr. Opin. Genet & Develop., 7:32-38. 74. Fitch, I., Dahmann, C , Surana, U . , Amon, A. , Nasmyth, K , Goetsch, L . , Byers, B. & Futcher, B. 1992. Characterization of four B-type cychn genes of the budding yeast Saccharomyces cerevisiae. Mol. Biol. Cell, 3:805—818. 75. Fleig, U . N . & Gould, K. L . 1991. Regulation of cdc2 activity in Schizosaccharomyces pombe: the role of phosphorylation. Semin. Cell Biol, 2:195—204. 76. Fraga, D., Yano, J., Reed, M . W., Chuang, R., Bell, W., Van Houten, J. L . & Hinrichsen, R. 1998. Introducing antisense oligodeoxynucleotides into Paramecium via electroporation. J. Euk. Microbiol. 45:582—588. 77. Frankel, J. 1962. The effects of heat, cold, and p-fluorophenylalanine on morphogenesis in synchronized Tetrahymena pyriformis GL. C. R. Trav. Lab. Carlsberg, 33:1—52. 78. Frankel, J. 1999. Cell biology of Tetrahymena thermophila. In Asai, D. & Forney, J. D. (ed.) Methods in Cell Biology. 62: 28-125. Academic Press, San Diego, CA. 79. Frankel, J., Jenkins, L . M . & DeBault, L . E. 1976. Causal relations among cell cycle processes in Tetrahymena pyriformis: An analysis employing temperature-sensitive mutants. J. Cell Biol, 71:242—260. 80. Frankel, J., Nelsen, E. M . & Jenkins, L . M . 1977. Mutations affecting cell division in Tetrahymena pyriformis, syngen 1. II. Phenotypes of single and double homozygotes. Dev. Biol, 58:255—275. 139 81. Frohman, M . A. 1990. R A C E : rapid amplification of cDNA ends. In: Snincky, J. J. & White, T. J. (ed.), PCR protocols. Academic Press, San Diego, CA. Pp. 28-38. 82. Fujishima, M . , Katsu, Y . , Ogawa, E., Sakimura, M . , Yamashita, M . & Nagahama, Y . 1992. Meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M -phase-promoting factor. / . Protozool, 39:683—690. 83. Gaertig, J. and Gorovsky, M . A. 1992. Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants. Proc. Natl. Acad. Sci. USA., 89:9196— 9200. 84. Gaertig, J., Thatcher, T. FL, Gu, L . & Gorovsky, M . A. 1994. Electroporation-mediated replacement of a positively and negatively selectable (3-tubulin gene in Tetrahymena thermophila. Proc Natl Acad Sci U S A., 91:4549—4553. 85. Galaktionov, K. & Beach, D. 1991. Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins. Cell, 67:1181-1194. 86. Gallant, P. & Nigg, E. A. 1992. Cyclin B2 undergoes cell cycle-dependent nuclear translocation and, when expressed as a non-destructible mutant, causes mitotic arrest in Hela cells. / . Cell Biol., 117:213—224. 87. Gautier, J., Norbury, C , Lohka, JVL, Nurse, P. & Mailer, J. 1988. Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+. Cell, 54:433-439. 88. Gerhart, J. C , Wu, M . & Kirschner, M . W. 1984. J. Cell Biol, 98: 1247—1255. 89. Girard, F., Strausfeld, U . , Fernandez, A. & Lamb, N . J. 1991. Cyclin A is required for the onset of D N A replication in mammalian fibroblasts. Cell, 67:1169—1179. 140 90. Glotzer, M , Murray, A. W. & Kirschner, M . W. 1991. Cyclin is degraded by the ubiquitin pathway. Nature, 349:132—138. 91. Grandin, N.& Reed, S. I. 1993. Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis. Mol. Cell. Biol., 13:2113-2125. 92. Greenwood, S. J., Sogin, M . L . & Lynn, D. H. 1991. Phylogenetic relationships within the class Oligohymenophorea, phylum Ciliophora, inferred from the complete small subunit rRNA gene sequences of Colpidium campylun, Glaucoma chattoni, and Opisthonecta henneguyi. J. Mol. Evol., 33:163—174. 93. Hadwiger, J. A. , Wittenberg, C., Mendenhall, M . D. & Reed, S. I. 1989. The Saccharomyces cerevisiae C K S l gene, a homolog of the Schizosaccharomyces pombe sucl+ gene, encodes a subunit of the Cdc28 protein kinase complex. Mol. Cell Biol, 9:2034-2041. 94. Haese, G. J. D., Walworth, N . , Carr, A. M . & Gould, K. L . 1995. The Weel protein kinase regulates T14 phosphorylation of fission yeast Cdc2. Mol. Biol. Cell, 6:371— 385. 95. Harnmerschmidt, B., Schlegel, M . , Lynn, D. FL, Leipe, D. D., Sogin, M . L . & Raikov, I. B. 1996. Insights into the evolution of nuclear dualism in the ciliates revealed by phylogenetic analysis of rRNA sequences. J. Euk. Microbiol, 43:225—230. 96. Hanks, S. K. , Quinn, A. M . & Hunter, T. 1988. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science, 241:42—52. 97. Hanon, G. J. and Beach, D. 1994. pl5mK4B is a potential effector of TGF-|3-induced cell cycle arrest. Nature, 371:257—261. 141 98. Harlow, E. & Lane, D. 1988. Antibodies, a laboratory manual. Cold Spring Harbor Laboratory Press New York. Pp. 473—510. 99. Harper, J. W., Adami, G. R., Wei, N . , Keyomarsi, K. & Elledge, S. J. 1993. The p21 Cdk-interacting protein Cipl is a potent inhibitor of G l cyclin-dependent kinases. Cell, 75:805-816. 100. Hartwell, L . H . & Weinert, T. A. 1989. Checkpoints: controls that ensure the order of cell cycle events. Science, 246:629—634. 101. Hartwell, L . H . 1974. Saccharomyces cerevisiae cell cycle. Bacterial. Rev., 38: 164—198. 102. Hartwell, L . H. , Culotti, J., Pringle, J. & Reid, B. J. 1974. Genetic control of the cell division cycle in yeast. Science, 183:46—51. 103. Hashimoto, N . & Kishimoto, T. 1988. Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation. Dev. Biol, 126: 242—252. 104. Hata, S., Kouchi, H. , Suzuka, I. & Ishii, T. 1991. Isolation and characterization of cDNA clones for plant cyclins. EMBO J., 10:2681-2688. 105. Hayles, J., Beach, D. H. , Durkacz, B. & Nurse, P. M . 1986. The fission yeast cell cycle control gene cdc2; isolation of a sequence Sucl that suppresses cdc2 mutant function. Mol. Gen. Genet., 202:291-293. 106. Hayles, J., Fisher, D., Woollard, A. & Nurse, P. 1994. Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34c d c 2-mitotic B cychn complex. Cell, 78:813-822. 142 107. Heald, R., Mcloughlin, M . & Mckeon, F. 1993. Human weel maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. Cell, 74:463-474. 108. Hershko, A . 1997. Roles of ubiquitin-mediated proteolysis in cell cycle control. Curr. Opin. Cell Biol, 9:788-799. 109. Higgins, D., Thompson, J. & Gibson, T. 1994. C L U S T A L W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22:4673— 4680. 110. Hinchchffe, E. H. , L i , C., Thompson, E. A. , Mailer, J. L . & Sluder, G. 1999. Requirement of Cdk2-Cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts. Science, 283:851—854. 111. Hindley, J. & Phear, G. A. 1984. Sequence of the cell division gene cdc2 from Schizosaccharomyces pombe: patterns of splicing and homology to protein kinase. Gene, 31:129-134. 112. Hindley, J., Phear, G., Stein, M . & Beach, D. 1987. Sucl+ encodes a predicted 13-kilodalton protein that is essential for cell viability and is directly involved in the division cycle of Schizosaccharomyces pombe. Mol. Cell. Biol, 7:504—511. 113. Hinrichsen, R. D., Fraga, D. & Reed, M . W. 1992. 3'-Modified antisense oligodeoxyribonucleotides complementary to calmodulin mRNA alter behavioral response in Paramecium. Proc. Nad. Acad. Sci. USA, 89:8601—8605. 143 114. Hirt, FL, Mink, M . , Pfosser, M . , Bogre, L. , Gyorgyey, J., Jonak, V. , Gartner, A. , Dudits, D. & Herberle-Bors, E. 1992. Alfalfa cyclins: differential expression during the cell cycle and in plant organs. Plant Cell, 4:1531—1538. 115. Hochstrasser, M . 1995. Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr. Opin. Cell Biol, 7:215—223. 116. Holz, G.G., Scherbaum, O.H., and Williams, N.E. 1957. The arrest of mitosis and stomatogenesis during temperature induction of synchronous division in Tetrahymena pyriformis, mating type I, variety I. Exp. Cell Res., 13: 618—621. 117. Home, M . C , Goolsby, G. L . , Donaldson,, K. L , Tran, D., Neubauer, M . & Wahl, A. F. 1996. Cyclin G l and cyclin G2 comprise a new family of cyclin with contrasting tissue-specific and cell cycle-regulated expression. J. Biol. Chem., 271:6050—6061. 118. Horowitz, S. & Gorovsky, M . A. 1985. An unusual genetic code in nuclear genes of Tetrahymena. Proc. Natl. Acad. Sci. USA, 82:2452-2455. 119. Huelsman D. A. , Gitz, D. L. & Pennock, D. G. 1992. Protein phosphorylation and the regulation of basal body microtubule organizing centers in Tetrahymena. Cytobios, 71: 37—50. 120. Inglis, R. J., Langan, T. A. , Matthews, H . R., Hardie, D. G. & Bradbury, E. M . 1976. Advance of mitosis by histone phosphokinase. Exp. Cell Res., 97:418—425. 121. Jackman, M . , Firth, M . & Pines, J. 1995. Human cyclins B l and B2 are localized to strikingly different structures: B l to microtubules, B2 primarily to the Golgi apparatus. EMBOJ., 14:1646-1654. 144 122. Jeffrey, P. D., Russo, A. A. , Polyak, K., Gibbs, E., Hurwitz, J., Massague, J. & Pavletich, N . P. 1995. Mechanism of C D K activation revealed by the structure of a cyclinA-CDK2 complex. Nature, 376:313-320. 123. John, P. C. L . , Sek, F. J. & Lee, M . G. 1989. A homolog of the cell cycle control protein p34 c d c 2 participates in the division cycle of Chlamydomonas, and a similar protein is detectable in higher plants and remote taxa. Plant Cell, 1:1185—1193. 124. Johnson, D. G. & Schneider-Broussard, R. 1998. Role of E2F in cell cycle control and cancer. Front. Biosci., 3:447—458. 125. Johnson, D. G. & Walker, C. L . 1999. Toxicol. Cyclins and cell cycle chechpoints. Annu. Rev. Pharmacol., 39:295-312. 126. Kaczanowska, J., Joachimiak, E., Buzanska, L. , Krawczynska, W., Wheatley, D. & Kaczanowski, A. 1999. Molecular subdivision of the cortex of dividing Tetrahymena is coupled with the formation of the fission zone. Dev. Biol., 212:150—164. 127. Kahn, R. W., Andersen, B. H . & Brunk, C. F. 1993. Transformation of Tetrahymena thermophila by microinjection of a foreign gene. Proc Natl Acad Sci USA., 90:9295—9299. 128. Kaldis, P., Sutton, A. & Solomon, M . J. 1996. The Cdk-activating kinase (CAK) from budding yeast. Cell, 86:553—564. 129. Kamb, A. , Gruis, N . A. , Weaver-Feldhaus, J., Liu, Q., Harshman, K. , Tavtigian, S. V. , Stockert, E. , Day, R. S., Johnson, B. E. & Skolnick, M . H . 1994. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 264:436—440. 130. Kaneda, M . & Hanson, E. D. 1974. Growth patterns and morphogenetic events in the cell cycle of Paramecium aurelia. In: Wagtendonk, W. J. van (ed.) Paramecium, a 145 current survey. Elsevier/North Holland Biomedical Press, New York Amsterdam, Pp. 219—262. 131. Karrer, K. M . 1986. The nuclear DNAs of holotrichous ciliates. In The molecular biology of ciliated protozoa. Academic Press. Pp. 85-110. 132. Kato, J., Matsushime, H. , Hiebert, S. W., Ewen, M . E. & Sherr, C. J. 1993. Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4. Genes Dev., 7:331—342. 133. Keryer, G., Davis, F. M . , Rao, P. N . & Beisson J. 1987. Protein phosphorylation and dynamics of cytoskeletal structures associated with basal bodies in Paramecium. Cell Motil. Cytoskel., 8: 44—54. 134. Kishimoto, T. & Kanatani, H. 1976. Cytoplasmic factor responsible for germinal vesicle breakdown and meiotic maturation in starfish oocytes. Nature, 260: 321—322. 135. Knighton, D. R., Zheng, J. H. , Ten, Eyck, L . F., Ashford, V . A. , Xuong, N . H . , Taylor, S. S. & Sowadski, J. M . Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science, 253:407—414. 136. Knoll, A. H . 1992. The early evolution of eukaryotes: A geological perspective. Science, 256:622—626. 137. Kobayashi, H. , Golsteyn, R., Poon, R., Stewart, E., Gannon, J., Minshull, J., Smith, R. & Hunt, T. 1991. Cyclins and their partners during Xenopus oocyte maturation. Cold Spring Harb Symp Quant Biol, 56:437-47. 138. Kobayashi, H. , Stewart, E., Poon, R., Adamczewski, J. P., Gannon, J. & Hunt, T. 1992. Identification of the domains in cyclin A required for binding to, and activation of, p34 c d c 2 and p32 c d k 2 protein kinase subunits. Mol. Biol. Cell, 3:1279-1294. 146 139. Koetsier, P. A. , Schorr, J. & Doerfler, W. 1993. A rapid optimized protocol for downward alkaline Southern blotting of DNA. BioTechniques, 15:260—261. 140. Krek, W. & Nigg, E. A . 1991. Differential phosphorylation of vertebrate p34 c d c 2 kinase at the Gl /S and G2/M transitions of the cell cycle: identification of major phosphorylation sites. EMBO J., 10:305—316. 141. Krek, W. & Nigg, E. A . 1992. Cell cycle regulation of vertebrate p34 c d c 2 activity: identification of Thrl61 as an essential in vivo phosphorylation site. New Biologist., 4:323-329. 142. Kubota, Y . , Mimura, S., Nishimoto, S., Takisawa, H . & Nojima, H . 1995. Identification of the yeast MCM3-related protein as a component of Xenopus D N A replication licensing factor. Cell, 81:601—609. 143. Labbe, J. C , Martinez, A. M . , Fesquet, D., Capony, J. P., Darbon, J. M . , Derancourt, J., Devault, A. Morin, N . , Cavadore, J. C. & Doree, M . 1994. p40 M O 1 5 associates with a p36 subunit and requires both nuclear translocation and Thrl76 phosphorylation to generate cdk-activating kinase activity in Xenopus oocytes. EMBO J., 13:5155-164. 144. Laemmli, U . K. 1970. Cleavage of structured proteins during the assembly of the head of the bacteriophage T 4 . Nature, 277:680—685. 145. Lee, M . G. & Nurse, P. 1987. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature, 327:31—35. 146. Lees, E. M . & Harlow, E. 1993. Sequences within the conserved cychn box of human cychn A are sufficient for binding to and activation of cdc2 kinase. Mol. Cell. Biol, 13:1194-1201. 147 147. Lehner, C. F. & O'Farrell, P. H . 1990. Drosophila cdc2 homologs: a functional homolog is coexpressed with a cognate variant. EMBO J., 9:3573—3581. 148. Lehner, C. F. & O'Farrell, P. H . 1989 Expression and function of Drosophila cyclin A during embryonic cell cycle progression. Cell, 56:957—968. 149. Lehner, C. F. & O'Farrell, P. H. 1990. The roles of Drosophila cyclins A and B in mitotic control. Cell, 61:535—547. 150. Lew, D. J., Dulic, V . & Reed, S. I. 1991. Isolation of three novel human cyclins by rescue of G l cyclin (Cln) function in yeast. Cell, 66:1197—1206. 151. Lew, J., Huang, Q. Q., Qi, Z., Winkfein, R. J., Aebersold, R., Hunt, T. & Wang, J. H . 1994. A brain-specific activator of cyclin-dependent kinase 5. Nature, 371:423— 426. 152. L i , J. J. & Herskowitz, I. 1993. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science, 262:1870—1874. 153. L i , J. J. 1995. D N A replication. Once, and only once. Curr. Biol, 5:472-475. 154. L i , Y . , Nichols, M . A. , Shay, J. W., Xiong, Y . 1994. Transcriptional repression of the D-type cyclin-dependent kinase inhibitor pl6 by the retinoblastoma susceptibility gene product pRb. Cancer Res., 54:6078—6082. 155. Lohia, A. & Samuelson, J. 1993. Cloning of the Ehcdc2 gene from Entamoeba histolytica encoding a protein kinase p34 c d c 2 homologue. Gene, 127:203—207. 156. Lohka M . & Masui, Y . 1983. Formation in vitro of sperm pronuclei and mitotic chromosome induced by amphibian ooplasmic components. Science, 220: 719—721. 148 157. Lohka, M . J. & Mailer, J. L . 1985. Induction of nuclear envelop breakdown, chromosome condensation and spindle formation in cell free-extracts. J. Cell Biol., 101: 518—523. 158. Lohka, M . J. & Mailer, J. L . 1987. Regulation of nuclear formation and breakdown in cell-free extracts of amphibian eggs. In Molecular Regulation of Nuclear Events in Mitosis and Meiosis (ed. R. A. Schlegel, M . K. Halleck & P. N . Rao), pp. 67-109. Orlando. Academic Press 159. Lohka, M . J., Hayes, M . K. & Mailer, J. L . 1988. Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acad. Sci. USA., 85:3009-3013. 160. Lopez-Girona, A. , Mondesert, O., Leatherwood, J. & Russell, P. 1998. Negative regulation of Cdcl8 D N A replication protein by Cdc2. Mol. Biol. Cell, 9:63—73. 161. Lorinze, A. & Reed, S. 1984. Primary structure homology between the product of yeast cell cycle control gene CDC28 and vertebrate oncogenes. Nature, 307:183—185. 162. Luo, Q., Michaelis, C. & Weeks, G. 1994. Overexpression of a truncated cychn B gene arrests Dictyostelium cell division during mitosis. J. Cell Sci., 107:3105—3114. 163. Maercker, C. & Lipps, H . J. 1994. A gene from the hypotrichous ciliate Stylonychia lemnae coding for a protein with homology to cyclin B. Gene, 141:145—146. 164. Makela, T. P., Tassan, J. P., Nigg, E. A. , Frutiger, S., Huges, G. J. & Weinberg, R. A. 1994. A cychn associated with the CDK-activating kinase M015. Nature, 371:254-257. 165. Mailer, J. L . 1993. On the importance of protein phosphorylation in cell cycle control. Mol. Cell Biochem., 127-128:267-281. 149 166. Marcote, M . J., Knighton, D. R., Basi, G., Sowadski, J. M . , Brambilla, P., Draetta, G. & Taylor, S. S. 1993. A three-dimensional model of the Cdc2 protein kinase: Localization of Cyclin- and Sue 1-binding regions and phosphorylation sites. Mol. Cell. Biol, 13:5122-5131. 167. Martin-Castellanos, C. & Moreno, S. 1996. Regualtion of G l progression in fission yeast by the ruml gene product. Prog. Cell Cycle Res., 2:29—35. 168. Martindale, D. W. 1989. Codon usage in Tetrahymena and other ciliates. J. Protozool, 36:29-34. 169. Masui, Y . & Markert, C. 1971. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zooi, 177:129—146. 170. Matsumoto, Y . H. , Yasuda, S., Mita, S., Marunouchi, T. & Yamada, M . 1980. Evidence for involvement of HI histone phosphorylation in chromosome condensation. Nature, 284:181—183. 171. McDonald, B. B. 1962. Synthesis of deoxyribonucleic acid by micro- and macronuclei of Tetrahymena pyriformis. J. Cell Biol., 13:193—203. CDC28 172. Mendenhall, M . D. 1993. An inhibitor of p34 protein kinase activity from Saccharomyces cerevisiae. Science, 259:216—219. 173. Meyerson, M . , Enders, G. H. , Wu, C.-L., Su, L . -K. , Gorka, C , Nelson, C , Harlow, E. & Tsai, L . - H . 1992. A family of human cdc2-related protein kinases. EMBO, J., 11:2909-2917. 174. Miake-Lye, R. & Kirschner, M . W. 1985. Induction of early mitotic events in a cell-free system. Cell, 41:165-175. 150 175. Michaelis, C. & Weeks, G. 1992. Isolation and characterization of a cdc2 cDNA fromDictyostelium discoideum. Biochim. Biophys. Acta., 1132:35—42. 176. Micklem, G., Rowley, A. , Harwood, J., Nasmyth, K. & Diffley, J. F. 1993. Yeast origin recognition complex is involved in D N A replication and transcriptional silencing. Nature, 366:87-89. 177. Minshull, J., Blow, J. J. & Hunt, T. 1989. Translation of cychn mRNA is necessary for extracts of activated Xenopus eggs to enter mitosis. Cell, 56:947—956. 178. Minshull, J., Golsteyn, R., Hill, C. S. & Hunt, T. 1990. The A- and B-type cychn associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBOJ., 9:2865-2875. 179. Moreno, S. & Nurse, P. 1994. Regulation of progression through the G l phase of the cell cycle by the ruml+ gene. Nature, 367:236—242. 180. Moreno, S., Hayles, J. & Nurse, P. 1989. Regulation of p34 c d c 2 protein kinase during mitosis. Cell, 58:361-372. 181. Morgan, D. O. 1995. Principles of C D K regulation. Nature, 374:131-134. 182. Nakamura, T., Sanokawa, R., Sasaki, Y . F., Ayusawa, D., Oishi, M . & Mori, N . 1995. Cychn I: a new cychn encoded by a gene ioslated from human brain. Exp. Cell Res., 221:534—542. 183. Nasmyth, K. 1993. Control of the yeast cell cycle by Cdc28 protein kinase. Curr. Opin. Cell Biol, 5: 166—179. 184. Nasmyth, K. 1996. At the heart of the budding yeast cell cycle. Trends. Genet., 12:405-412. 151 185. Nigg, E. A. 1995. Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. BioEassays, 17:471-480. 186. Ninomiya-tsuji, J., Nomoto, S., Yasuda, H. , Reed, S. & Matsumoto, K. 1991. Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation. Proc. Natl. Acad. Sci. USA, 88:9006-9010. 187. Nobori, T., Miura, K. , Wu, D. J., Lois, A. , Takabayashi, K. & Carson, D. A. 1994. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature, 368:753-756. 188. Noda, A. , Ning, Y. , Venable, S. R , Pereira-Smith, O. M . & Smith, J. R. 1994. Cloning of senescent cell-derived inhibitors of D N A synthesis using an expression screen. Exp. Cell Res. 211:90-98. 189. Norbury, C , Blow, J. & Nurse, P. 1991. Regulatory phosphorylation of the p34 c d c 2 protein kinase in vertebrates. EMBO J., 10:3321—3329. 190. Nurse, P. & Bissett, Y . 1981. Gene required in G l for commitment to cell cycle and in G2 for control of mitosis in fission yeast. Nature, 292: 558—560. 191. Nurse, P. 1975. Geneic control of cell size at cell division in yeast. Nature, 256:547— 551. 192. Nurse, P. 1994. Ordering S phase and M phase in the cell cycle. Cell, 79: 547—550. 193. Nurse, P. Thuriaux, P. & Nasmyth, K. 1976. Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet., 146: 167—178. 194. Nurse, P., & Bissett, Y . 1981. Gene required in G l for commitment to cell cycle and in G2 for control of mitosis in fission yeast. Nature, 292:558—560. 152 195. Ohtsubo, M . , Theodoras, A . M . , Schumacher, J., Roberts, J. M . & Pagano, M . 1995. Human cychn E, a nuclear protein essential for the Gl-to-S phase transition. Mol. Cell Biol, 15:2612-2624. 196. Pagano, M . , Durst, M . , Joswig, S., Draetta, G. & Jansen-Durr, P. 1992. Binding of the human E2F transcription factor to the retinoblastoma protein but not to cychn A is abolished in HPV-16-immortahzed cells. Oncogene, 7:1681—1686. 197. Pagano, M . , Pepperkok, R., Lukas, J., Baldin, V. , Ansorge, W., Bartek, J. & Draetta, G. 1992. Cychn A is required at two points in the human cell cycle. EMBO J., 11:961-971. 198. Page, R. D. M . 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences, 12:357—358. 199. Pardee, A. 1974. A restriction point for control of normal animal cell proliferation. Proc. Natl. Acad. Sci. USA., 71:1286—1290. 200. Pasternak, J. 1967. Differential gene activity in Paramecium aurelia. J. Exp. Zool., 165:395-418. 201. Peeper, D. S., Parker, L . L. , Ewen, M . E., Toebes, M . , Hall, F. L . , Xu , M . , Zantema, A. , Eb, A. J. & Piwnica-Worms, H . 1993. A- and B-type cyclins differentially modulate substrate specificity of cyclin-cdk complexes. EMBO J., 12:1947—1954. 202. Peter, M . & Herskowitz, I. 1994a. Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell, 79:181—184. 203. Peter, M . & Herskowitz, I. 1994b. Direct inhibition of the yeast cyclin-dependent kinase Cdc28-Cln by Farl . Science, 265:1228—1231. 153 204. Peter, M . , Gartner, A. , Horecka, J., Ammerer, G. & Herskowitz, I. 1993. FAR1 links the signal transduction pathway to the cell cycle machinery in yeast. Cell, 73:747—760. 205. Peter, M . , Nakagawa, J., Doree, M . , Labbe, J. C. & Nigg, E. A . 1990. In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell, 61:591-602. 206. Pines, J. & Hunter, T. 1989. Isolation of a human cychn cDNA: evidence for cychn mRNA and protein regulation in the cell cycle and for interaction with p34 c d c 2. Cell, 58:833-846. 207. Pines, J. & Hunter, T. 1990. Human cychn A is adenovirus ElA-associated protein p60 and behaves differently from cychn B. Nature, 346:760—763. 208. Pines, J. & Hunter, T. 1991. Human cychn A and B I are differentially located in the cell and undergo cell cycle -dependent nuclear transport. J. Cell Biol., 115:1—17. 209. Pines, J. 1995. Cyclins and cyclin-dependent kinases: a biochemical view. Biochem. J., 308:697-711. 210. Pines, J. 1996. Cychn from sea urchins to Helas: making the human cell cycle. Colworth Medal Lecture. Biochem. Soc. Trans., 24:15—33. 211. Pines, J. and Hunter, T. 1989. Isolation of a human cychn cDNA: evidence for cychn mRNA and protein regulation in the cell cycle and for interaction with p34 c d c 2. Cell, 58:833-846. 212. Polyak, K., Lee, M . H , Erdjument-Bromage, H , Koff, A. , Roberts, J. M . , Tempst, P. & Massague, J. 1994. Cloning of p27Kipl, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell, 78:59—66. 154 213. Poon, R. Y . , Yamashita, K., Adamczewski, J. P., Hunt, T. & Shuttleworth, J. 1993. The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34 c* 2. EMBO J., 12:3123-3132. 214. Preer, J. R., Preer, L . B. , Rudman, B. M . & Barnett, A. J. 1985. Deviation from the universal code shown by the gene for surface protein 51A in Paramecium. Nature, 314:188-190. 215. Prescott, D. M . 1994. The D N A of ciliated protozoa. Microbiol. Rev., 58:233-267. 216. Rao, P. N . & Johnson, R. T. 1970. Mammalian cell fusion: studies on the regulation of D N A synthesis and mitosis. Nature, 225:159—164. 217. Rasmussen, C. D. & Berger, J. D. 1982. Downward regulation of cell size in Paramecium tetraurelia: effects of increased cell size, with or without increased D N A content, on the cell cycle. J. Cell Sci., 57:315-329. 218. Rasmussen, C. D., Berger, J. D. & Ching, A. L . 1986. Effects of increased cell mass and altered gene dosage on the timing of initiation of macronuclear D N A synthesis in Paramecium tetraurelia. Exp. Cell Res., 165:53—62. 219. Rasmussen, L . & Zeuthen, E. 1962. Cell division and protein synthesis in Tetrahymena, as studied with p-fluorophenylalanine. C. R. Trav. Lab. Carlsberg, 32:333—358. 220. Reed, S. I., Hadwiger, J.A. & Lorincz, A. T. 1985. Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28. Proc Natl Acad Sci U S A., 82:4055-4059. 221. Richardson, H . E. , Stueland, C. S., Thomas, J., Russell, P. & Reed, S. I. 1990. Human cDNAs encoding homologs of the small p34 c d c 2 8 / c d c 2-associated protein in 155 Saccharomyces cerevisiae and Schizosaccharomyces pombe. Genes Dev., .4:1332— 1334. 222. Richardson, H . E., Wittenberg, C , Cross, F. & Reed, S. I. 1989. An essential G l function for cyclin-like proteins in yeast. Cell, 59:1127—1133. 223. Richardson, H. , Lew, D. J., Henze, M . Sugimoto, K. & Reed, S. I. 1992. Cyclin B homo logs in Saccharomyces cerevisiae function in S phase and in G2. Genes Dev., 6:2021—2034. 224. Rickert, P., Seghezzi, W., Shannhan, F., Cho, H. & Lees, E. 1996. Cyclin C/CDK8 is a novel CTD kinase associated with RNA polymerase II. Oncogene, 12:2631—2640. 225. Riley, D. E. , Campell, L . A. , Puolakkainen, M . & Krieger, J. N . 1993. Trichomonas vaginalis and early evolving D N A and protein sequences of the CDC2/28 protein kinase family. Mol. Microbiol., 8:517—519. 226. Rogers, S., Wells, R. & Rechsteiner, M . 1986. Amino acid sequences common to rapid degraded proteins: the PEST hypothesis. Science, 234:364—368. 227. Rosenthal, E. T., Hunt, T. & Ruderman, J. V . 1980. Selective translation of mRNA controls the pattern of protein synthesis during early development of the surf clam, Spisula solidissima. Cell, 20:487—494. 228. Ross-MacDonald, P. B. , Graeser, R., Kappes, B. & Williamson, D. H . 1994. Isolation and expression of a gene specifying a cdc2-like gene protein kinase from the human malaria parasite Plasmodium falciparum. Eur. J. Biochem., 220:693—701. 229. Roth, S. Y . , Collini, M . P., Draetta, G., Beach, D. & Allis, C. D. 1991. A cdc2-We kinase phosphorylates histone HI in the amitotic macronucleus of Tetrahymena. EMBOJ., 10:2069-2075. 156 230. Roy, R., Adamczewski, J. P., Seroz, T., Vermeulen, W., Tassan, J. P., Schaeffer, L . , Nigg, E. A. , Hoeijmakers, J. H . & Egly, J. M . 1994. The M O 15 cell cycle kinase is associated with the TFIIH transcription-DNA repair factor. Cell, 79:1093—1101. 231. Ruiz, F., Beisson, J., Rossier, J. & Dupuis-Williams, P. 1998a. Basal body duplication in Paramecium requires y-tubulin. Curr. Biol., 9:43—46. 232. Ruiz, F., Vayssie, L . , Klotz, C , Sperling, L . & Madeddu, L . 1998b. Homology-dependent gene silencing in Paramecium. Mol. Biol. Cell, 9:931—943. 233. Russell, C. B. , Fraga, D. & Hinrichsen, R. D. 1994. Extremely short 20-33 nucleotide introns are the standard length in Paramecium tetraurelia. Nucleic Acids Res., 22:1221-1225. 234. Russo, A. A. , Jeffrey, P. D. & Pavletich, N . P. 1996. Structural basis of cyclin-dependent kinase activation by phosphorylation. Nat. Struct. Biol, 3:696—700. 235. Sambrook, J., Fritsch, E. F. & Maniatis, T. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor laboratory Press, New York. 236. Satterwhite, L . L. , Lohka, M . J., Wilson, K. L . , Scherson, T. Y . , Cisek, L . J., Corden, J. L . & Pollard, T. D. 1992. Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc: a mechanism for the timing of cytokinesis. J. Cell Biol., 118:595-605. 237. Schafer, E. & Cleffmann, G. 1982. Division and growth kinetics of the division mutant conical of Tetrahymena. A contribution to the regulation of generation time. Exp. Cell Res., 137:277—284. 238. Scherbaum, O. & Zeuthen, E. 1954. Induction of synchronous cell division in mass cultures of Tetrahymena pyriformis. Exp. Cell Res. 6:221-227. 157 239. Schwob, E. & Nasmyth, K. 1993. CLB5 and CLB6, a new pair of B cyclins involved in D N A replication in Saccharomyces cerevisiae. Genes Dev., 7:1160—1175. 240. Schwob, E., Bohm, T., Mendenhall, M . D. & Nasmyth, K. 1994. The B-type cychn kinase inhibitor p40 s / c 7 controls the G l to S transition in S. cerevisiae. Cell, 79:233— 244. 241. Serrano, M . , Hannon, G. J. & Beach, D. 1993. A new regulatory motif in cell-cycle control causing specific inhibition of cychnD/CDK4. Nature, 366:704—707. 242. Sharp, P .M. , Cowe, E., Higgins, D. G., Shields, D. C , Wolfe, K. H . & Wright, F. 1988. Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity. Nucl. Acid. Res., 16:8207—8211. 243. Shen, X . , Yu, L. , Weir, J. W. & Gorovsky, M . A. 1995. Linker histones are not essential and affect chromatin condensation in vivo. Cell, 82:47—56. 244. Sherr, C. J. 1993. Mammalian G l cyclins. Cell, 73:1059-1065 245. Simanis, V . & Nurse, P. 1986. The cell cycle control gene cdc2 of fission yeast encodes a protein kinase potentially regulated by phosphorylation. Cell, 45: 261—268. 246. Smith, D. B. & Johnson, K. S. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusion with glutathione S-transferase. Gene, 67:31— 40. 247. Smythe, C. & Newport, J. W. 1992. Couphng of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34" / c 2. Cell, 68:787-797. 158 248. Sogin, M . L . 1991. Early evolution and the origin of eukaryotes. Curr. Opin. Genet. Dev., 1:457—463. 249. Solomon, M . J. 1993. Activation of the various cychn/cdc2 protein kinases. Curr. Opin. Cell Biol, 5:180-186. 250. Solomon, M . J., Harper, J. W. & Shuttleworth, J. 1993. C A K , the p34 a / e 2 activating kinase, contains a protein identical or closely related to p40 M O 1 5 . EMBO J., 12:3133— 3142. 251. Solomon, M . J., Lee, T. & Kirschner, M . W. 1992. Role of phosphorylation in p34 e d c 2 activation: identification of an activating kinase. Mol. Biol. Cell, 3:13—27. 252. Sonneborn, T. M . 1970. Methods in Paramecium research. Methods Cell Physiol, 4:241-339. 253. Sperhng L. , Keryer, G., Ruiz, F. & Beisson J. 1991. Cortical morphogenesis in Paramecium: a transcellular wave of protein phosphorylation involved in ciliary rootlet disassembly. Dev. Biol, 148: 205—218. 254. Standart, N . M . , Bray, S. J., George, E. L. , Hunt, T. & Ruderman, J. V . 1985. The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs. J Cell Biol, 100:1968—1976. 255. Standart, N . , Minshull, J., Pines, J. & Hunt, T. 1987. Cychn synthesis, modification and destruction during meiotic maturation of the starfish oocyte. Dev. Biol, 124:248— 254. 256. Stern, B. & Nurse, P. 1996. A quantitative model for the cdc2 control of S phase and mitosis in fission yeast. Trends Genet., 12:345—350. 159 257. Stuart, D. & Wittenberg, C. 1995. CLN3, not positive feedback, determines the timing of CLN2 transcription in cycling cells. Genes Dev., 9:2780—2794. 258. Sweet, M . T. & Allis, C. D. 1993. Phosphorylation of linker histones by cAMP-dependent protein kinase in mitotic micronuclei of Tetrahymena. Chromosma, 102:637-647 259. Swenson, K. I., Farrell, K. M . & Ruderman, J. V . 1986. The clam embryo protein cychn A indices entry into M-phase and the resumption of meiosis in Xenopus oocytes. Cell, 47:861-870. 260. Tang, L . , Adl, M . S. & Berger, J. D. 1997. A cdc2-related kinase is associated with macronuclear D N A synthesis in Paramecium tetraurelia. J. Euk. Microbiol., 44:269— 275. 261. Tang, L. , Pelech, S. L . & Berger, J. D. 1994. A cdc2-like kinase associated with commitment to division in Paramecium tetraurelia. J. Euk. Microbiol., 41:381—387. 262. Tang, L . , Pelech, S. L . & Berger, J. D. 1995. Isolation of the cell cycle control gene cdc2 from Paramecium tetraurelia. Biochim. Biophys. Acta., 1265:161—167. 263. Tang, Y . & Reed, S. I. 1993. The Cdk-associated protein Cksl functions both in G l and G2 in Saccharomyces cerevisiae. Genes Dev., 7:822—832. 264. Tassan, J. P., Schultz, S. J., Bartek, J. & Nigg, E. A. 1994. Cell cycle analysis of the activity, subcellular localization, and subunit composition of human C A K ( C D K -activating kinase). 7. Cell Biol, 127:467'-478. 265. Thuret, J. Y . , Valay, J. G., Faye, G. & Mann, C. 1996. C iv l (CAK in vivo), a novel Cdk-activating kinase. Cell, 86:565—576. 160 266. Tondravi, M . and Yao, M-C. 1986. Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes. Proc. Natl. Acad. Sci. USA., 83:4369—4373. 267. Toyoshima, H . and Hunter, T. 1994. P27, a novel inhibitor of G l cyclin-cdk protein kinase activity, is related to p21. Cell, 78:67—74. 268. Tsai, L . H. , Delalle, I., Caviness, V. S. Jr, Chae, T.& Harlow, E. 1994 p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature, 371:419-423. 269. Tucker, J. B. , Beisson, J., Roche, D. L . J. & Cohen, J. 1980. Microtubules and control of macronuclear 'amitosis' in Paramecium. J. Cell Sci., 44:135—151. 270. Tyers, M . & Futcher, B. 1993. Farl and Fus3 link the mating pheromone signal transduction pathway to three Gl-phase Cdc28 kinase complexes. Mol. Cell Biol., 13:5659-5669. 271. Valay, J. G., Simon, M . & Faye, G. 1993. The kin28 protein kinase is associated with a cyclin in Saccharomyces cerevisiae. J. Mol. Biol., 234:307—310. 272. Wang, J., Chenivesse, X . , Henglein, B. & Brechot, C. 1990. Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma. Nature, 343:555—557. 273. Wang, S., Nakashima, S., Saka, H. , Numata, O., Fujiu, K. & Nozawa, Y . 1998. Molecular cloning and cell-cycle-dependent expression of a novel N I M A (never-in-mitosis in Aspergillus nidulans)-re\ated protein kinase (TpNrk) in Tetrahymena cells. Biochem. J., 334:197—203. 274. Waseem, N . H. , Labib, K., Nurse, P. & Lane, D. P. 1992. Isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein PCNA. EMBO J., 11:5111-5120. 161 275. Wei, P., Garber, M . E. , Fang, S. M . , Fischer, W. H . & Jones, K. A. 1998. A novel CDK9-associated C-type cyclin interacts directly with FJIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell, 92:451^162. 276. Wei, Y . , Yu, L. , Browen, J., Gorovsky, M . & Allis, C. D. 1999. Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell, 97:99-109. 277. Westendorf, J. M . , Swenson, K. I. & Ruderman, J. V . 1989. The role of cyclin B in meiosis I. J. Cell Biol, 108:1431—1444. 278. Williams, N . E. , Honts, J. E. & Kaczanowska, J. 1990. The formation of basal body domains in the menbrane skeleton of Tetrahymena. Development. 109:935—942. 279. Williams, N . E . & Macey, M . G. 1991. Is cyclin Zeuthen's "division protein"?. Exp. Cell Res., 197:137-139. 280. Williams, N . E. & Nelsen, E. M . 1997. Ffsp70 and Hsp90 homologs are associated with tubulin in heter-oligomeric complexes, cilia and the cortex of Tetrahymena. J. Cell Sci., 110:1665-1672. 281. Wolfe, J. 1976. G l arrest and the division/conjugation decision in Tetrahymena. Dev. Biol, 54:116—426. 282. Won, K. A. & Reed, S. I. 1996. Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin-dependent degradation of cyclin E. EMBO J., 15:4182-4193. 283. Wu, M . , Allis, D., Sweet, M . , Cook, R., Thatcher, T. and Gorovsky, M . A. 1994. Four distinct and unusual linker proteins in a mitotically dividing nucleus are derived 162 from a 71-Kilodalton polyprotein, lack p34c c sites, and contain protein kinase A sites. Mol. Cell. Biol, 14:10—20. 284. Xiong, Y . , Hannon, G. J., Zhang, H. , Casso, D., Kobayashi, R. & Beach, D. 1993. P21 is a universal inhibitor of cychn kinases. Nature, 366:701—704. 285. Xiong, Y . , Menninger, J., Beach, D. & Ward, D. C. 1992. Molecular cloning and chromosomal mapping of CCND genes encoding human D-type cyclins. Genomics, 13:575—584. 286. Xiong, Y . , Zhang, H. , Casso, D., Kobayashi, R. & Beach, D. 1993. P21 is a universal inhibitor of cychn kinases. Nature, 366:701—704. 287. Ye, X . S. & Osmani, S. A. 1997. Regulation of p34 c d c 2/cycknB HI and N I M A kinases during the G2/M transition and checkpoint responses in Aspergillus nidulans. Prog. Cell Cycle Res., 3:221—232. 288. Zhang, H . & Berger, J. D. 1999. A novel member of the cyclin-dependent kinase family in Paramecium tetraurelia. J. Euk. Microbiol, 46:482—491. 289. Zhang, H , Adl, S. M . & Berger, J. D. 1999. Two distinct classes of mitotic cyclins homologues, Cycl and Cyc2, are involved in cell cycle regulation in the cihate Paramecium tetraurelia. J. Euk. Microbiol, 46:585—596. 290. Zindy, F., Lamas, E., Chenivesse, X . , Sobczak, J., Wang, J., Fesquet, D., Henglein, B. & Brechot, C. 1992. Cychn A is required in S phase in normal epithelial cells. Biochem. Biophys. Res. Commun., 182:1144—1154. 163 A P P E N D I X I. DESCRIPTION OF OLIGONUCLEOTIDES Oligo 1: 5 A T G A / C G A G C A / T A T T / A T / C T A / G G / A T A / T G A - 3 ' Oligo 2: 5 A T T / C T C T / C T C A / G T A T / C T T T / A G / C A / T T / A G C - 3 ' Degenerate sense and antisense primers based on mitotic cyclin box consensus sequences M R A I L V and A S K Y E E I , respectively, for cloning CYC1 and CYC2 from P. tetraurelia, and cyclin-like sequences from Blepharisma intermedium, Sterkiella histriomuscorum, Colpoda sp. and Tetrahymena thermophila. Oligo 3: 5 ' - G G A / T G A A / G G G A / T A C A / T T A T / C G G - 3 ' Oligo 4: 5 ' -G/ATTT/CTA/GA/TGGC/TTTA/TAA/GG/ATC-3 ' Degenerate sense and antisense primers based on the two conserved regions of known Cdk sequences G E G T Y G (protein kinase domain I) and D L K P Q N (domain VI), respectively, for cloning CDK2 from P. tetraurelia and CDK1 from T. thermophila. Ada: 5 ' - G A C T C G A G T C G A C A T C G - 3 ' dTn: 5 ' -GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3 ' Universal primers for 3' and 5' RACEs Oligo 5: 5 ' - T A T G A T G A A T C A A G A T T A - 3 ' Oligo 6: 5 ' - T A G T T A G A T A G A A T G C A A - 3 ' Outer and internal primers for 3' R A C E to clone 3' end sequence of PtCDK2. 164 Oligo 7: 5 A T A G T T T A A G G C T T G T A T C A T - 3 ' Specific reverse transcription primer for 5' R A C E of PtCDKl. Oligo 8: 5 - T T G C A A T T C T A T C T A A C T A - 3 ' Oligo 9. 5 '-T A A T C T T G A T T C A T C AT A-3 ' Outer and internal primers for 5' R A C E to clone 5' end sequence of PtCDKl. Oligo 10: 5 ' - T C T T T G G C T C A A G T C A C - 3 ' Oligo 11: 5 ' -TAGGTGTGGCAGCCTTG-3 ' Outer and internal primers for 3' R A C E to clone 3' end sequence of PtCYClb. Oligo 12: 5 ' - C A C T A G A A T G G C T C T C A T C T T - 3 ' Specific reverse transcription primer for 5' R A C E of PtCYClb. Oligo 13: 5 ' - C C T G C A G C A G G T G T C A T A T A C T A - 3 ' Oligo 14: 5 ' - T C C A T C A G C A A G T G T T - 3 ' Outer and internal primers for 5' R A C E to clone 5' end sequence of PtCYClb. Oligo 15: 5 ' - A A C T T A T A T G T T A C A A C T T A T - 3 ' Oligo 16: 5 ' - A G T T G G A G T A G C T T C A T T A - 3 ' Outer and internal primers for 3' R A C E to clone 3' end sequence of PtCYCl. Oligo 17: 5 ' - T A T A A A T A A T G A A G C T A C T C C A - 3 ' Specific reverse transcription primer for 5 ' R A C E of PtCYC2. Oligo 18: 5 ' -GTTATTATCTGGTTGTAGTCTT-3 ' Oligo 19. 5 ' -AT A A G T T G T A A C AT AT A A G - 3 ' Outer and internal primers for 5' R A C E to clone 5' end sequence of PtCYC2. Oligo 20. 5 ' -CTTCGTCCTTACCAAAT-3 ' Oligo 21: 5 - T G C C A C A A G C C G T A G A A T - 3 ' Outer and internal primers for 3' R A C E to clone 3' end sequence of TtCDKl. Oligo 22: 5 ' - G T C T T G A T A G G A A C A C C A - 3 ' Specific reverse transcription primer for 5' R A C E of TtCDKl. Oligo 23: 5 ' -ATTCTACGGCTGTGGCA-3 ' Oligo 24: 5 ' - A T T T G G T A A G G A C G A A G - 3 ' Outer and internal primers for 5' R A C E to clone 5' end sequence of TtCDKl. A P P E N D I X II. NUCLEOTIDE A N D AMINO ACID SEQUENCES OF P. TETRAURELIA CYC1 GENE ISOFORMS T A A T A A T T T A T T T A A T A T T A A C A G A CYClb T A G - G A CYCla. A T G A T C A T C G A A A A T C A A A G A T G T T T T G G G A A G G A A A T A G C A A A T T C A A C T T T G C A T C A A T C C A A G G A G CyclA M I I E N Q R C F G K E I A N S T L H Q S K E CyclB _ _ _ - _ - _ _ _ - - - - - - - s v - - - - -CYClb T - - G A G C - - T T G - - C A - T - C A CYCla A T A G G C A T A A T C G T G G A A A A A C A T A A A A A A C C T T T C T C C A T A A T A C C A A A A G T T T T T G C A A T G A G T T T G CyclA I G I I V E K H K K P F S I I P K V F A M S L CyclB - - M - - - - - - E - - - - - - - - - T T - -CYClb T A - A C C C - - A T A - - C CYCla G A T G A T A A A G A A A A T A A A C T G T T T A G A A G A G A A T C A G A A A A A T T C T A A A T T G A G A T A G A A A C C G A A A A G CyclA D D K E N K L F R R E S E K F Q I E I E T E K CyclB - - - _ _ _ _ - - - - - - - i p - - - - i D -CYClb - - T A C A C C T A A - - A - - A GG A — T T - G G CYCla A G C A A G G A T G T T A A A A A T C C T T A A A A T G T T G A G T T A T A T T C C A A T G A G A T C T T A C A A C A C C T A C T G A T T CyclA S K D V K N P Q N V R L Y S N E I L Q H L L I CyclB - - E H L - - - K - E - - - D - - - - - - - M CYClb A C A - - T T T A - - C T C - T - - G CYCla G A G G A G A A T A A A T A T A C A A T T A A C T A G T A C A T G A C A C C T G A G T A A C A G C C T G A T A T C A A C A T A A A A A T G CyclA E E N K Y T I N Q Y M T P E Q Q P D I N I K M CyclB - - - - - - - - - - - - - - - - - - - - L - -CYClb C A G C T - - G A T - - T CYCla A G A G C T A T T C T T G T G G A T T G G T T A A T T G A T G T T C A T G C C A A A T T C G A G T T G A A G G A T G A A A C A C T C T A C CyclA R A I L V D W L I D V H A K F E L K D E T L Y CyclB - _ _ - - - _ - v - - - - - - K - - - - - - -CYClb - - C T - - A - - A - - C T T - - T C A G G CYCla A T T A C A A T C T C T T T G A T T G A T C G A T A C T T G G C T C T G G C T C A A G T C A C A A G A A T G A G A T T A C A G T T A G T A CyclA I T I S L I D R Y L A L A Q V T R M R L Q L V CyclB _ - _ - _ _ _ _ _ _ s - - - - - - - K - - - -167 CYClb A T C — C A A A — A CYCla GGTGTGGCTGCCCTGTTTATAGCTTGTAAGTACGAAGAAATCTACCCTCCTGCTTTGAAGGATTTCGTT CyclA G V A A L F I A C K Y E E I Y P P A L K D F V CyclB -CYClb C C A T C - T C G - A CYCla TACATAACAGATAATGCGTATGTGAAAAGTGACGTTTTGGAAATGGAAGGTTTAATGTTATAAGCCTTA CyclA Y I T D N A Y V K S D V L E M E G L M L Q A L CyclB - - - - - - - - - - - - - - - - - - - - - - -CYClb C T A C - - C T T G - - G G - C A CYCla AATTTCAATATATGCAATCCCACTGCTTATTAATTCTTACAAAAATACTCAACCAATTTAGATCCGAAG CyclA N F N I C N P T A Y Q F L Q K Y S T N L D P K CyclB - - _ - - _ _ _ _ - _ _ _ _ _ _ _ _ D - - - -CYClb A A A — A CYCla GATAAGGCGTTAGCTTAATATATACTGGAATTGGCTTTAGTTGAATATAAATTTATTATATATAAGCCT CyclA D K A L A Q Y I L E L V L V E Y K F I I Y K P CyclB N - - - - - - - - - - - - - - - - - - - - - -CYClb T CT C C G — T — G C G TA T CYCla TCTTAAATTGTCCAATCTGTTATATTTTTAGTTAATAAGATTAGAACACCCACTTATAAAACACCGAAC CyclA S Q I V Q S V I F L V N K I R T P T Y K T P N CyclB - L - A - - - - - - - - - - - - - - H - - Q -CYClb G C G - - - T — G A T T - G CYCla GAGAATCAATTAAAGCCTTGTGCTAAAGAATTATGCACATTACTTTAAACAGCAGATCTAAGTTCCCTA CyclA E N Q L K P C A K E L C T L L Q T A D L S S L CyclB - - - - - - - - - - - - - - - - - - - - N - -CYClb C C - A A CYCla TAAGCAGTAAGGAAGAAATTCAATGCTTCTAAATTTTTTGAAGTTTCGAGAATTAAAGTAGAAAAAACA CyclA Q A V R K K F N A S K F F E V S R I K V E K T CyclB _ _ _ _ _ _ _ _ _ T _ _ _ _ _ _ _ _ _ _ _ _ _ CYClb C CYCla AACAAATGATTGTAGTTTTAAATGTCTTGTTTCTTTGACTTTCACTCAAATTTTCCAAAAAAAAAAAAA CyclA N K * CyclB - N * 168 CYClb CYCla AAAAA < u I—I H O H W H I pq O l < H O >. U a: 00 " l-H P H P H O u H I I H I l >H I I « I I O I < < I I H I J En l l I I I I I I I EH Q I I I < O P < U CO H CO PM Q EH o CJ P > H fc fc ^ & P a i w J l u fc i i a o s >H i s < CO EH E H CO I CM I i s a > U EH < H I > £ CO P fc I I S P E H J I I < E H > a I i*5 i-3 I I S H H j a i > i < i i i i i i fc 1 1 I I i PM p 1 H H H w W « i I O Q p 1 > > F> > > < p P H P w PM I I I i 1 Pi CM I I I i 1 PM >H 1 H 1 I I I 1 i i 1 1 >* H LO ro LO LO o LO ro LO o CN CN CN LO o rH rH Ch ro rH [> r> LO cn LO ro ro rH LO rH ro rH rH rH CN rH CN rH rH CN CN CN CN CN CN ro ro ro ro < rH < rH < rH rH CN < < < < • rH CN < < < < • rH CN < < < • U U CJ CJ CJ CJ ti U CJ CJ U CJ CJ ti CJ u CJ CJ CJ CJ ti >1 >1 >H >H >H >H 0 >1 > i >* 0 > i >1 >H >H >H 0 u u u CJ CJ CJ U u u u u u CJ o CJ u CJ u U u O 4J -P w rH -U 4J w rH Cfl JJ 4-> Cfl rH Cfl CM CM CO p X! *! CM CM CO p X a < CM CM CO p X <«" W I I >H I I tf I I U I C O < I I H I I I P j i a C O C O < I E H H > J I I H H < O £ > a PM tf i i i i i i Q tf CM O tf l > m o a H H H P < > W EH P < > > O H W pq i a tf tf J a a i i i i i i a Q ex pi a i i i tf i >H H W Q • > I P H > C O J I pa i p i H J >H I a i < i j i <1 C O tf J P W P Q I I I I I Q Q P EH l l H co a En < I I I I I I I a H a p i I P a E H I I a p i tf tf tf tf i co i i rt; i a i i E H E H C O En EHI r-rH I | I i I ' ' I ' l-M M-l l-M l-M HH LO ro O CTl rH CN a> UD o CN CN Ch CTl o rH rH o 00 O r - !> i n CN yo ro ro rH o 00 CN rH rH CM CM rH CN rH rH CN ro CN CN CN CN ro ^ CN ro < rH rH < rH rH < rH rH rH CN PQ PQ PQ PQ • rH CN pq pq pq pq • rH CN PQ PQ PQ PQ • o U u U o u CJ u U U U U a u u U O u u fi >1 >i >H >H >H 0 >1 >1 I* >H 0 >1 • > 1 >H 0 u U u U C J U o o u U U U U o u u u O u u o 4-) -U w rH w 4J 4J to rH w - P - P w rH w CM CM C O P Xi n CM CM C O P X ffl CM CM C O P X! CQ A P P E N D I X IV. SINGLE A N D TRIPLE LETTER CODES OF AMINO ACIDS Amino acid Single-letter code Triple-letter code Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamic acid E Glu Glutamine Q Gin Glycine G Gly Histindine H His Isoleucine I He Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val 172 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0089702/manifest

Comment

Related Items