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A CDC2-related kinase from Paramecium tetraurelia Tang, Liren 1995

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A CDC2-RELATED K INASE F R O M PARAMECIUM TETRAURELIA by LIREN T A N G B.Sc , Hunan Normal University, China, 1983 M . S c , Chinese Academy of Sciences, 1986 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF T H E REQUIREMENTS FOR THE D E G R E E 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 September, 1995 © L I R E N T A N G , 1995 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 ^ 0 O L-O^ V The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ABSTRACT Cell division in higher eukaryotes is mainly controlled by p34cdc2, a ser-ine/threonine protein kinase, and/or related kinases, and by other components of these kinase complexes. I present evidence that CDC2-like kinases also occur in the ciliate Paramecium tetraurelia. The protein encoded by the isolated Paramecium cdc2 homo-logue did not bind to p l3 s u < : ; , was localized in the macronucleus, its associated kinase ac-tivity was high at the initiation of macronuclear DNA synthesis, and it was active as a monomer. To study the relationship between the cellular and molecular events of cell cycle regulation, synchronous cultures are essential. However, in Paramecium, the only reliable technique for obtaining synchronous cell populations has been hand-selection of dividing cells. This technique is only useful for small samples and impractical for biochemical analysis. In this thesis, centrifugal ehxtriation, which fractionates the cell population on the basis of sedimentation properties with minimal perturbation of metabolic function, was applied to the ciliate Paramecium tetraurelia. Only the smallest cell fractions were well synchronized and exhibited synchrony and cell cycle duration equivalent to hand-selected samples. These small cell fractions consisted of a highly synchronous G l cell population, which was easily obtained by this technique and used for all subsequent molecular and biochemical analysis. With a combination of various polymerase chain reaction (PCR) techniques, a cdc2 homologous sequence was isolated from Paramecium which is referred to as cdc2PtA. The genomic Paramecium cdc2PtA gene contained two short introns near the 5'-end. The corresponding amino acid sequence exhibited about 50 % identity to the cdc2 proteins of other eukaryotes. The Paramecium cdc2PtA gene-encoded protein was 11 amino acids longer than that of Schizosaccharomyces pombe. It had most of the catalytic sites re-quired for CDC2 kinase activity, especially those phosphorylation sites which regulate U l CDC2 kinase activity in other organisms. There was one amino acid change in the highly conserved PSTAIRE region and other changes in regions which are required for interac-tion with other regulatory proteins, especially the pl3*" e / binding sites. Southern blot analysis as well as isolation of a second incomplete cDNA sequence from the 3'-end indi-cated that Paramecium has multiple cdc2 genes. Northern blotting results showed that the Paramecium cdc2PtA gene was much more strongly expressed in actively dividing cells than in starved stationary phase cells in which cdc2PtA mRNA was almost undetectable. There was no significant change in cdc2PtA mRNA level throughout the vegetative cell cycle. Polyclonal antibodies were produced against both a synthetic peptide from the C-tenninal region and a GST-CDC2PTA fusion protein which contained a third of the Paramecium cdc2PtA protein from the N-terminal region. Both antibodies recognized a 36 kDa polypeptide on Western blots. The antibodies did not cross-react with protein extracts from Tetrahymena or S. pombe, nor with the Paramecium 34 kDa polypeptide which was detected by anti-PSTATJRE antibody. The Paramecium CDC2PTA protein level decreased slightly when cells entered stationary phase and was invariant throughout the cell cycle, similar to its transcription pattern. Indirect immunofluorescence results showed that Paramecium CDC2PTA protein was located in the macronucleus, but not observed in the micronuclei or cytoplasm Upon starvation, the strength of the fluorescence signal in the macronucleus dropped slightly, consistent with the result from Western blotting. Native Paramecium CDC2PTA kinase was immunoprecipitated with the Parame-cium CDC2PTA specific antibody. The precipitated CDC2PTA kinase phosphorylated both bovine histone H I and casein in vitro, but not retinoblastoma (Rb) protein. Using histone H I as substrate, CDC2PTA kinase activity was assayed in the ehitriation synchro-nized samples. Histone H I kinase activity was high during the early stages of the cell cycle and reached a peak at around 2.5 hr after ehitriation, which corresponded approximately to the time of the initiation of macronuclear D N A synthesis. This suggests that the isolated iv Paramecium CDC2PTA kinase may be associated with the regulation of macronuclear D N A synthesis. When Paramecium extracts were probed with anti-PSTAIRE antibody, two polypeptides were detected. The major one migrated at 36 kDa was apparently recognized by anti-CDC2PTA antibody. The minor one migrated at the same position as S. pombe p3Acdc2 protein. Only the faster migrating one showed affinity for p 13™c7 protein. The phosphotransferase activity of the p l 3 i U C / precipitable protein was very low at early stages and increased at around 1.5 hr before cell division. This kinase activity increase corre-sponded to the point of commitment to division in Paramecium. Immunoprecipitation results showed that Paramecium CDC2PTA kinase occurred principally as monomers. This was further confirmed by glycerol density gradient centrifu-gation and gel filtration. These monomers were active as a histone H I kinase in vitro. These observations indicate that isolated Paramecium CDC2-like kinase differs from typi-cal CDC2 kinases in terms of interaction with and regulation by other cell cycle regulatory components. V T A B L E O F C O N T E N T S A B S T R A C T i i T A B L E O F C O N T E N T S v LIST O F T A B L E S viii LIST O F F I G U R E S ix LIST O F A B B R E V I A T I O N S xii A C K N O W L E D G M E N T S xv G E N E R A L I N T R O D U C T I O N 1 A. The CDC2/CDC28-like Protein Kinase Family 2 B. C D K Regulatory Factors 6 C. Substrates for CDKs 11 D. The Significance of Cell Cycle Regulation Studies 13 E. Paramecium and Its Cell Cycle 15 F. Organization of the Thesis 18 M A T E R I A L S A N D M E T H O D S 20 A. Materials 20 B. Stocks and Cell Culture..... 20 C. Centrifugal Ehitriation 21 D. Labeling of Paramecium with [3H]-Thymidine 22 E. D N A and R N A Isolation 23 F. PCR Amplification ...23 G. Subcloning of PCR Products 24 FL Isolation and Sequencing of Genomic and cDNA cdc2 Gene 25 vi I. D N A Probe Preparation 26 J. Southern Blot Analysis 27 K. Northern Blot Analysis 28 L. Relative Level of cdc2PtA Transcripts by RT-PCR 29 M . Preparation of Paramecium Protein Extracts 30 N . Expression in E. coli and GST-Fusion Protein Purification 31 O. Production and Purification of Antibodies 32 P. Protein Electrophoresis and finmunoblotting 33 Q. Immunofluorescence 33 R Metabolic Labeling of Paramecium Cells 34 S. S. pombe Cells and Protein Extraction 34 T. pl3* u c / Precipitation : 35 U. Immunoprecipitation 35 V . Glycerol Density Gradient Centrifugation 36 W. Gel Filtration s 37 X . Kinase Activity Assay 37 C H A P T E R O N E : S Y N C H R O N I Z A T I O N O F PARAMECIUM C E L L S B Y C E N -T R I F U G A L E L U T R I A T I O N 39 1.1 INTRODUCTION 39 1.2 RESULTS 40 1.3 DISCUSSION 43 C H A P T E R T W O : I S O L A T I O N O F T H E C E L L C Y C L E C O N T R O L G E N E CDC2 F R O M PARAMECIUM TETRAURELIA 46 2.1 INTRODUCTION 46 2.2 RESULTS 47 2.2.1 Isolation of a ccfc2-like Sequence from Paramecium by PCR 47 2.2.2 Isolation and Characterization of Genomic and cDNA cdc2PtA 48 2.2.3 Similarity of CDC2PTA to Other CDC2-like Proteins 51 2.2.4 Paramecium Has Multiple cdc2 Genes 54 2.3 DISCUSSION 57 C H A P T E R T H R E E : G E N E E X P R E S S I O N O F CDC2PTA D U R I N G T H E C E L L C Y C L E 61 3.1 INTRODUCTION : 61 3.2 RESULTS 63 3.2.1 Cdc2PtA Transcripts are Abundant in Actively Dividing Cells 63 3.2.2 CDC2PTA Protein is Invariant Throughout the Cell Cycle..... 69 3.2.3 CDC2PTA Protein Localizes Mainly in Macronucleus 73 3.3 DISCUSSION 75 C H A P T E R F O U R : C H A R A C T E R I Z A T I O N O F C D C 2 P T A K I N A S E 79 4.1 INTRODUCTION .79 4.2 RESULTS 80 4.2.1 CDC2PTA Protein does not associate with P13™c/ 80 4.2.2 CDC2PTA Kinase Activity Throughout the Cell Cycle 83 4.2.3 CDC2PTA May Have Kinase Activity as Monomer 90 4.3 DISCUSSION 98 G E N E R A L DISCUSSION A N D P E R S P E C T I V E S 105 S U M M A R Y I l l L I T E R A T U R E C I T E D 115 A P P E N D I C E S 127 viii List of Tables Table 1. Table 2. Properties of CDKs and related kinases identified in mammalian systems. Possible substrates for cyclin-dependent kinases ix List of Figures Fig. 1-1. A schematic illustration of the involvement of vertebrate CDK/cyclin complexes in cell cycle progression. Fig. 1-2. CDC2 regulation by protein kinases and phosphatases in S. pombe. Fig. 1-3. Paramecium Cell Cycle Control Model Fig. 1-1. Synchronization of Paramecium cells by centrifugal elutriation. Fig. 1-2. Fractionation of Paramecium cells by centrifugal elutriation. Fig. 2-1. Nucleotide and deduced amino acid sequences of Paramecium cdc2 gene, cdc2PtA. Fig. 2-2. PCR amplification from the two intron regions. Fig. 2-3. Alignment of the predicted amino acid sequence of CDC2PTA with CDC2 or CDC2-like kinases of various species. Fig. 2-4. Comparison of the two different Paramecium cdc2-like sequences (cdc2PtA and cdc2PtB). Fig. 2-5, Southern hybridization of Paramecium tetraurelia genomic D N A with cdc2PtA. Fig. 3-1. Northern blotting analysis o£cdc2PtA gene expression from starved and expo-nentially growing cells. A . EtMdium bromide-stained gel of total RNA. B . Northern hybridization with cdc2PtA sequence. Fig. 3-2. Expression of cdc2PtA gene during the vegetative cell cycle by Northern blot-ting. A . Division kinetics. B. Northern blotting analysis. C. Quantitaiton of the Northern blotting. Fig. 3-3. Expression ofcdc2PtA by RT-PCR A . Determination of linearity between RT-PCR products and R N A input. B. Detection of cdc2PtA gene expression dur-X ing the cell cycle by R T - P C R C. Quantitative determination of the linearity of R N A imputs and PCR products. D. Cell division kinetics and quantitaion of gene expression during the cell cycle by RT-P CR Fig. 3-4. Immunodetection by CDC2PTA specific antibodies. A . Anti-GST-CDC2PTA fusion protein antibody. B. Anti-peptide antibody. Fig. 3-5. The CDG2PTA protein levels during the cell cycle. A . Cell division kinetics. B. Protein levels as a function of cell cycle stages. C. Protein level of CDC2PTA between starved and exponentially growing cells. Fig. 3-6. Immunofluorescence localization of CDC2PTA. B and D: Immunostaining with CDC2PTA antibody. A and C: D N A staining with DAPI. Fig. 4-1. Pl3sucI binding analysis. A . Paramecium protein extracts immunoblotted with CDC2PTA antibody. B. S. pombe extracts. C. Tetrahymena extracts. D. Paramecium extracts immunoblotted with anti-PSTATRE antibody. Fig. 4-2. In vitro kinase assay. Fig. 4-3. Endogenous substrate for CDC2PTA. Fig. 4-4. Histone H kinase assay as a function of cell cycle stage. Fig. 4-5. CDC2PTA associated histone H I kinase activity between exponentially growing and starved cells. Fig. 4-6. Immunoprecipitation analysis using [35S]-labeled proteins. Fig. 4-7. Sedimentation of [35S]-labeled proteins by glycerol density gradient centrifuga-tion. Fig. 4-8. Sedimentation by glycerol density gradient centrifugation. A . Immunodetection of Paramecium proteins. B. Immunodetection of S. pombe proteins. C. B S A standard. D. Kinase activity. r xi Fig. 4-9. Gel-filtration chromatography of Paramecium cell lysate. A . Immunoblotting with anti-PSTAIRE antibody. B. Histone H I kinase activities of gel-filtered proteins. xii List of Abbreviations aa amino acid ATP adenosine triphosphate bp base pair B S A bovine serum albumin cAMP cyclic adenosine 3',5'-monophosphate C D K cyclin dependent kinase CDKI cyclin dependent kinase inhibitor cDNA complementary deoxyribonucleic acid cpm Courie per minute DAPI 4',6-diamidino-2-pheny indole DEPC diethyl pyrocarbonate D N A deoxyribonucleic acid dNTP deoxyribonucleotide triphosphates DTT 1,4-dithiothreitol E D T A emylenediaminetetraacetate E G T A ethylene glycol-bis-N,N,N',N'-tetraacetate g gram GST glutathione S-transferase hr hour JDS initiation of D N A synthesis ffTG isopropyl 3-D-thiogalactopyranoside uCi micro Curie u,g micro gram X U l Ml micro liter u M micro molar M molar miti minute mg milligram ml milliliter MPF maturation promoting factor mRNA messenger ribonucleic acid ng nanogram n M nanomolar PBS phosphate buffered saline PCD point of comittment to division PCR polymerase chain reaction PMSF phenylmethylsulfonyl fluoride R A C E rapid amplification of cDNA ends R A G E rapid amplification of genomic ends Rb retinoblastoma R N A ribonucleic acid rRNA ribosomal ribonucleic acid rpm revolution per minute SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate poryacrylamide gel electrophoresis T E M E D N , N , N ' , N ' -tetramethylemylenediamme Tris tris(hydroxymethyl) aminomethane TBS Tris buffered saline U unit X-gal 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside xiv Amino acid and nucleotide sequences are indicated by the single letter code (Appendices I and JJ). Genes are written in italic (for example cdc2). Proteins are indicated by capital letters (for example, CDC2). S. pombe genes are shown in lower italicized cases (for ex-ample, cdc25). S. cerevisiae genes are shown in upper italicized cases (fro example, CDC28). X V Acknowledgments My deepest appreciation and gratitude are expressed to Dr. Berger, who not only provided me with an excellent research project, but also his guidance and advice. His pa-tience and encouragement throughout the course of this research project was the biggest support. I am greatly indebted to Dr. Pelech for creative advice, support and his kindly al-lowing me to use his equipment and reagents. Without his invaluable assistance, this the-sis would have been impossible. I am also very grateful to other members of my supervisory committee, Dr. Grigliatti, Dr. Brock, Dr. Matsuuchi and Dr. Moerman, for their time, efforts, and critical contribution at every stage of this study. Warmest thanks are given to Dr. Daya-Makin for her support and counsel, and for teaching me biochemical techniques at the beginning of this study. David Charest and Harry Paddon from Dr. Pelech's laboratory are also acknowledged for technical assis-tance. I thank Longjun Dai and Xiwu Zhou for their help in making graphs and scintilla-tion counting. A l l members of the Berger laboratory were very special. I would like to thank Sina A d l , Gordon Chua, Monika Havelka, Hong Zhang for thier enjoyable working environ-ment, friendship, excellent scientific discussions, and patience with language problems. The Natural Science and Engineering Council of Canada (NSERC) is thanked for financial support for research. Last but not least, I wish to thank my wife, Caixia, for her tremendous emotional support, encouragement, and endurance throughout my years as a student. 1 G E N E R A L I N T R O D U C T I O N The cell cycle encompasses the processes of D N A replication, mitosis, pre-fission morphogenesis and cytokinesis that leads to the production of two daughter cells from a single cell. This cycle is typically divided into four phases. The events of D N A replication (S phase) and nuclear division (mitosis, M phase) are separated by gaps of varying length called G l and G2. A l l cell types undergo some version of this basic cycle, although details of regulation and the length of the gap phases differ. The normal well-ordered cell division process depends on the proper coordination of cell cycle events. This coordination is mainly produced by the regulation of two critical transitions. The first is called "START" in yeasts and is a point of commitment to ini-tiation of D N A synthesis that occurs in the late G l interval The second is the G2 /M transition, which gates cells into mitosis and cell division. In the eukaryotic cell divi-sion cycle, many of the important decisions including these two transitions are deter-mined and regulated by a growing family of cyclin-dependent protein kinases (CDKs) (reviewed by Morgan, 1995; Pelech etal, 1990; Solomon, 1993). The pathway leading to the full activation of these kinases is quite complex. The C D K catalytic activity is regulated by phosphorylation/dephosphorylation as well as by the formation of com-plexes with other regulatory components. In general, each C D K needs to bind to one of a family of broadly-related regulatory subunits called cyclins in order to become active as a kinase. Both the association of each cychh-CDK pair, and the activity of the result-ing cyclin-CDK complex are closely regulated by phosphorylation/dephosphorylation of the C D K subunit. Once activated, different cyclin-CDK complexes are responsible for phosphorylating the various substrates that are required for such diverse cell cycle events as the initiation of D N A synthesis (IDS), chromosome condensation, and nuclear envelope breakdown (reviewed by Nigg, 1993; Solomon, 1993). 2 A. The CDC2/CDC28-Like Protein Kinase Family The first identified CDK, CDC2ICDC28, now named CDK1 in higher eukaryo-tes, was originally identified as the 36 kDa protein product of the CDC28 gene from Saccharomyces cerevisiae and later as the 34 kDa product of the cdc2 gene of Schi-zosaccharomyces pombe. Related genes have been isolated from diverse species includ-ing algae, protozoa, fungi, plants, and animals. In fission yeast and Aspergillus, CDC2 is the only C D K identified; in budding yeast, the major CDK, CDC28, has been recently joined by other members of the family, KIN 28 kinase and PH085 kinase (Kaffinan et al., 1994; Measday, et al., 1994; Espinoza et al., 1994; Valay et al, 1993). Both of cdc2 and CDC28 kinases are associated with a cyclin-like protein. However, in mul-ticellular organisms, especially vertebrate animals, it appears that the cell cycle is regulated by a family of C D K members closely related to CDC2 kinase. Based primarily on sequence-homology screening, more than ten cDNAs have been isolated from mammalian cells which encode proteins that are closely related to cdc2/CDC28 (Table 1). Among these, seven including cdc2, encode proteins with molecular masses of ap-proximately 32-36 kDa (40-42 kDa for CDK6 and CDK7). These protein kinases have been shown, or are suggested, to associate with cyclin, and therefore have been re-ferred to as cyclin-dependent kinases (CDK1-CDK7) (Meyerson et al, 1992; Tassan et al, 1994). Of these, only three, cdkl, cdk2 and cdk3, are able to substitute for a de-fective cdc2 gene when introduced into S. pombe cells (Meyerson, et al, 1992). Our understanding of C D K structure and function is largely based on the studies of the ma-jor C D K (CDK1) in yeast and vertebrates, and CDK2 in vertebrates. 3 Table 1 Properties of CDKs and Related Kinases Identified in Mammalian Cells* Name PSTAIRE Size Associated Identity to CDC2 Motif kDa aa Cyclin(s) % CDC2(CDK1) PSTAIRE 34 297 A & B 100 CDK2 PSTAIRE 33 298 A , D & E 65 CDK3 PSTAIRE 36 305 ? 66 CDK4(PSKJ3) PISTVRE 36 303 D & E 44 CDK5 PSSALRE 31 291 D & p 3 5 57 CDK6 PLSTTRE 40 326 D 47 CDK7(CAK) N R T A L R E 42 346 H 44 PCTAIRE(l-3) PCTAIRE 51-58 451- 523 ? 51-55 p58 PITSLRE 58 434 ? 42 K K I A L R E K K I A L R E 43 358 ? 43 *: This table is modified from review articles of Pines (1993); Solomon (1994); Lew and Wang (1995): Grana and Reddy (1995). For further references, see these reviews. 4 Although S. cerevisiae modulates its cell cycle chiefly at G l / S , whereas S. pombe regulates its cell cycle progression chiefly at G2/M, genetic studies have demonstrated that cdc2/CDC28 is required both for "START" and for the G2/M transition. Absence of the cdc2/cdc28 function in either yeast arrests the cell cycle at both G l / S and G2/M transitions (Forsburg and Nurse, 1991; Hartwell et al, 1973; Nurse and Bisset, 1981; Reed, 1980; Reed and Wittenberg, 1990; Sudbury et al, 1980), The cdc2/CDC28 func-tion does not appear to be regulated at the transcriptional or translation level, since neither the cdc2/CDC28 transcripts or their protein levels change during the cell cycle or on entry into stationary phase (Moreno et al, 1989; Surana et al, 1991). However, in vitro kinase activity associated with cdc2/CDC28 oscillates during the vegetative cell cycle (Moreno et al, 1989; Reed and Wittenberg, 1990; Wittenberg and Reed, 1988). The vertebrate CDK1 is mainly needed for entry into mitosis, and CDK2 ap-pears to function in controlling Gl/S-phase events. At present, the role of CDK3 in the human cell cycle is not clear. A dominant negative mutant of cdk3 arrests cell cycle during G l phase, which suggests that it may be required for G l events (Van den Heu-vel and Harlow, 1993). Three other CDKs, CDK4, CDK5 and CDK6, are found in association with the D-type cyclins and may be involved in the regulation of G l phase too (Bates et al, 1994; Matsushime et al, 1992; Matsushime et al, 1994; Xiong et al, 1992; Fig. 1-1). CDK5 is also thought to be required for neural cell differentiation (recently reviewed by Lew and Wang, 1995). CDK7 activates both CDC2 and CDK2, but neither its expression nor its kinase activity changes during the cell cycle (Tassan et al, 1994). The cell cycle roles of other CDC2-related kinases are currently unknown. 5 Fig. 1-1. A schematic illustration of the involvement of vertebrate CDK/cyclin com-plexes and other regulatory components in cell cycle progression. The specific CDK/cyclin complexes that function at various stages of the cell cycle are shown along with the various regulatory proteins that control their phosphotransferase activity. See text for details. 6 B. CDK Regulatory Factors B-l : CDK Activation by Cyclin Binding Genetic and biochemical analysis in yeast and vertebrates have identified several genes that regulate cell cycle through C D K kinases. Monomeric CDKs are unphospho-rylated and have no kinase activity. A l l known CDKs need to bind to a member of the cyclin family in order to become active kinases (Table 1). In particular, the periodicity of C D K 1 and CDK2 kinase activity is dependent on their cell cycle-dependent associa-tion with various cyclins, which vary in their abundance throughout the cell cycle. In yeasts, cdc2ICDC28 kinase is associated with many distinct cyclins at different points of the cell cycle, whereas human CDKs react with relatively few. In addition, a single mammalian cyclin can sometimes partner with multiple CDKs (reviewed by Grana and Reddy, 1995; Fig. 1-1; Table 1). Originally, cyclins were defined as proteins whose lev-els oscillate during the cell cycle. They are now more accurately defined as members of a family of structually related proteins that bind and activate C D K catalytic subunits. Homology among cyclins is often limited to a relatively conserved domain of about 100 amino acids, the cyclin box, which is responsible for C D K binding and activation (Zheng and Ruderman, 1993). Six types of cyclins have been identified from mammalian cells (reviewed by Grana and Reddy, 1995). The A-type cyclins associate with both CDK1 and CDK2, and play a role in both S phase and mitosis. There are two B-type cyclins and their ex-pression peaks at G2 phase. Both B l and B2 cyclins bind predominantly to CDK1 and activate CDK1 at mitosis. The C-type cyclin is expressed maximally at G l phase and its physiological C D K partner is still obscure. D-type cyclins appear to function at the G0/G1 transition, and associate with CDK2, CDK4 and CDK6. There are at least 3 D-type cyclins that are differentially expressed in various cell types, usually at the G0/G1 transition or in G l phase, and are turned over rapidly before S phase. The E-type cy-7 clins are expressed at the G l / S transition and associate with CDK2 and CDK4 (Table 1 and Fig. 1-1). Cyclins are thought to contain regions that target C D K t o specific sub-strates or subcellular location. B-2: CDK Activation by Phosphorylation CDC2/CDK2 activity is also subject to additional regulation by phosphoryla-tion/dephosphorylation. Binding of CDK1/CDK2 to cyclin induces phosphorylation at three sites: Thr-14, Tyr-15, and Thr-167 in S. pombe cdc2 (Thr-161 in human). Thr-161/167 phosphorylation is required for catalytic activity (reviewed by Solomon, 1993). In higher eukaryotes, this phosphorylation is carried out by a CDK-activating kinase (CAK), itself a member of the ccfc2-related kinase family (CDK7) (Holly et al, 1995; Solomon et al, 1993; Solomon, 1994; Tassan etal, 1994). C A K is composed of a complex of at least one regulatory subunit (p37) which is called cyclin H even though it shares limited homology with the cyclin family (Fisher and Morgan, 1994; Makela et al, 1994). The CDK7/cyclin H complex can readily phosphorylate CDK1, CDK2 and CDK4 complexed with various cyclins. Phosphorylation of Thr-161/167 tends to rise and fall in parallel with cyclin binding during the cell cycle. However, neither the CDK7 phosphotransferase activity nor its protein level appears to fluctuate at different stages of the cell cycle. B-3: CDK Inhibition by Phosphorylation The cyclin-CDK complex can be inhibited by phosphorylation at the conserved Thr-14/Tyr-15 pair (Solomon, 1993). These phosphorylations are particularly impor-tant in the control of CDC2 activity at mitosis. The CDC2-mitotic cyclin complexes are maintained in an inactive state before G2/M transition, until Thr-14/Tyr-15 dephospho-rylation at the end of G2 activates CDC2. A Y15F mutation results in premature acti-8 vation of cdc2 in S. pombe and as a result, cells expressing the mutant divides at a re-duced size (Gould and Nurse, 1989). Tyr-15 phosphorylation of cdc2 in S. pombe is governed by a protein kinase encoded by wee J and its related kinase mkl (Lundgren et al, 1991; McGowan and Russell 1993; Fig. 1-2). The weel kinase also phosphorylates Thr-14 of S. pombe cdc2 (Flaese et al., 1995). Thr-14 inhibitory kinase has been re-cently detected in Xenopus and human cells (Atherton-Fessler et al., 1994). Based on an in vitro assay system in HeLa cells and Xenopus, both Thr-14 and Tyr-15 specific kinase activities have been found to be high throughout interphase and diminish coinci-dent with entry into mitosis (Atherton-Fessler et al., 1994). hi S. pombe, weel activity also declines during mitosis due to its phosphorylation by another 67 kDa protein kinase encoded by the niml gene (Fig. 1-2). Higher eukaryotic homologs of N T M l have not been found. The final step of activation of CDKl-cyclin complex is the removal of inhibitory phosphate from Tyr-15 (and also Thr-14 in vertebrates) by the protein tyrosine phos-phatase cdc25 which was also originally identified in S. pombe. Cdc25 activity increases during mitosis because of its increased phosphorylation. During mitosis, the kinase re-sponsible for this phosphorylation is activated and the corresponding phosphatase is inhibited. The human cdc25 homolog has been cloned. Both S. pombe and human CDC25 have been shown to be capable of directly removing phosphate from both Thr-14 and Tyr-15 (Gautier et al, 1991; Hoffmann et al, 1993; Millar and RusseL 1992; Sebastian etal, 1993). Therefore, in order to initiate the entry of cells into mitosis ( M phase), CDC2 must form a complex with a cyclin and must be phosphorylated at Thr-161/Thr-167 by C A K But to ultimately trigger entry into M phase, it has to be dephosphorylated at Thr-14 and Tyr-15 by cdc25 (Fig. 1-2). 9 G2 Phase M Phase Fig. 1-2. Cdc2 regulation by protein kinases and phospatases in S. pombe. During in-terphase, newly synthesized cdcl3 (cyclin B homologue) binds to cdc2 inducing its phosphorylation on 3 residues. Phosphorylation of Thr-14 and Tyr-15 by weellmikl kinases is inhibitory, whereas phosphorylation of Thr-167 activates cdc2. The inhibitory phosphorylations are reversed by cdc25 phosphatase and dephosphorylation of these sites induces mitosis. Exit from mitosis occurs with degradation of cdc 13 and dephos-phorylation of Thr-161 by an unknown phosphatase. 10 B-4: CDK Inhibition by Inhibitory Subunits CDKs are also modulated by a diverse family of inhibitory proteins, termed the CKI, that bind and inactivate CDK-cyclin complexes. At present,more than ten mem-bers of this family have been clearly identified (reviewed by Peter and Herskowitz, 1994; Elledge and Harper, 1994; Grana and Reddy, 1995; Lee et al., 1995; Fig. 1-1). Two CKIs from S. cerevisiae, FAR] and p40 (SICJ/SDB25), inhibit CDC28ICLN(Gl cyclin) and CDC28ICLB (S. cerevisiae mitotic cyclin) complexes, respectively. A third yeast C K I (PH081) inhibits the activity of the PHO85IPHO80 complex (Morgan, 1995). Among the four major mammalian CKIs, a 21 kDa protein (p21/WAFl/CAP20/SDI/CIPl) binds to CDK1, CDK2 and CDK4. Its related 27 kDa protein (KD?l/p27) associates preferentially with CDK2/cyclin E and CDK4/cyclin D complexes. The other two closely related CKIs ( p i d 0 * 4 and p lS 1 1 ^ 4 8 ) are specific for CDK4- and C D K 6-cyclin complexes (Elledge and Harper, 1994; Serrano et al, 1995). The C K I inhibitory mechanisms remain to be resolved. Most CKIs bind tightly to Thr 160/161-phosphorylated CDK-cyclin complexes and directly inhibit kinase ac-tivity. In many cases (FAR1, p40, p21 and p27), CKIs are phosphorylated by the CDK-cyclin complex (Elledge and Harper, 1994; Peter and Herskowitz, 1994). Many CKIs apparently recognize CDK-cyclin complexes rather than C D K monomers. On the other hand, p l6 associates with CDK4 monomers in vivo, suggesting it may block cyclin binding (Serrano et al, 1995). S. pombe pl3sucI is another protein that interacts with CDKs. Homologues have been identified from human and S. cerevisiae (Hartwiger et al, 1989; Richardson et al, 1990). Its role in cell cycle control is not clear. It has been proposed that the p I3suel gene inhibits the activation of cdc2 or promotes the inactivation of cdc2, because dele-tion of the sue J gene causes cells to arrest in mitosis and overexpression leads to a de-11 delay of the entry into mitosis (Brizuela et al., 1987; Hayles et al., 1986; Hindley et al., 1987; Moreno et al., 1989). C . Substrates for C D K There is no shortage of potential candidate substrates for C D K kinases in vitro (reviewed by Nigg, 1993; Table 2). However, there are comparatively very few pro-teins that are definitely phosphorylated by CDKs in vivo and whose phosphorylation is known to be relevant to cell cycle progression have been identified. Why should the C D K kinases phosphorylate so many different substrates? A possible explanation is that the consensus motif for the C D K phosphorylation site (mainly based on p34" / c 2), Ser/Thr-Pro-X-Z (where X is a polar amino acid and Z is generally a basic amino acid), is found in many proteins. How different CDKs in vertebrates and a single C D K in yeast bring about various events during the cell cycle by phosphorylating different sub-strates is unclear. The functional consequences of phosphorylation of different substrates by CDKs have been a subject of speculation. As listed in Table 2, the potential conse-quences resulting from phosphorylation of various proteins can be linked to almost all the events occuring during the cell cycle, e.g., transcriptional activation, D N A replica-tion, mitotic division, cytokinesis. The only direct evidence for functional consequences is the correlation between CDC2 activation and phosphorylation of the lamin compo-nents of the nuclear envelope. The phosphorylation site of lamin in vivo is identical to these phosphorylation sites by p34 c < / c 2 in vitro, and the phosphorylation of these sites increases during mitosis when y34cdc2 phosphotransferase activity is high. The direct phosphorylation of lamin by p34 c < / c 2 leads to nuclear disaasembfy (reviewed by Nigg, 1992). 12 Table 2 Possible Substrates for Cyclin-Dependent Kinases* Targets for CDKs Timing Potential Consequences Chromatin Associated Proteins: Histone H I Nucleolin High-mobility group proteins(HMGl,Y,Pl) S and M phases early M phase M phase chromatin condensation nuclear disassembly chromatin decondensation Cytoskeleton Proteins: Nuclear lamins Vimentin Neurofilament H Myosin light chain Caldesmon early M phase M phase M phase M phase M phase nuclear envelope breakdown intermediate filament disassembly dissociation from microtubules cytokinesis inhibition mitotic microfilament reorganization Transcription Factors: SW15 c-Myb c-Fos/Jun Oct-1 S G 2 & M phases M phase nuclear localization repression ? ? Protein Kinases: p6(T c Casein Kinase JJ P150"6' c-Mos M phase phase ? phosphorylation of further targets phosphorylation of further targets abolishing D N A binding of c-Abl D N A Replication Factors: SV40T antigen RPA (replication fac-tor) D N A polymerase a S phase S & G2 phases D N A replication initiation stimulating D N A unwinding and replica-tion ? 13 Tumor Suppressors: p53 G l phase pRb(retinoblastoma) G l phase stimulating D N A binding of p53 Miscellaneous Proteins: Casein ? R A B l/RAB4(RAS-like M phase GTP binding protein) Lamin B receptor M phase EF- ly (elongation fac- M phase tor) R N A polymerase II M phase dissociation from membranes dissociation of lamins from a membrane protein (p58) decreasing translation increasing transcription CDC25 phosphatase M phase activation of CDC25 Cyclin B M phase regulation of CDC2 activity *: This table is modified from the review article by Nigg (1993). For futher references, see Nigg (1993). D. The Significance of Cell Cycle Regulation Studies The ability of a cell to reproduce by cell division is one of the most fundamental processes of all living matter. Therefore, a thorough understanding of the events in-volved in normal cell division is crucial to understanding transformed cell growth. The ultimate goal of the cell cycle studies will be a link between altered cell cycle function and tumorigenesis. Cancer is a disease characterized by loss of cellular growth control. As such, it is not surprising that the molecular machinery of the cell cycle is involved in tumorigenesis. Recent discoveries have brought several cell cycle regulators into focus as factors in human cancer (reviewed by Kamb, 1995; Hartwell and Kastan, 1994; Hunter and Pines, 1994), especially cyclin D and C D K inhibitors. 14 In mammalian cells, the regulation of the START point and entry into the cell division cycle from stationary phase are most closely linked to D type cyclins and then-associated partner CDKs, primarily CDK4 and in some cells, CDK6 also. Overexpres-sion of certain cyclins, especially those of the D class, contribute to cell transformation (Hunter and Pines, 1994). The connection between cyclin D and cancer are strength-ened by the evidence that cyclin D is fundamental to cell cycle regulation of the first identified tumor suppressor retinoblastoma (Rb) protein. The connection between the Rb protein and cell cycle regulation was recently reviewed by Weinberg (1995). The main role of Rb protein is to act as a signal transducer connecting the cell cycle clock with the transcriptional niachinery. The cyclin D-CDK4 complexes only phosphorylate Rb and transcriptional factor E2F-1 in vitro (Matsushime et al., 1994; Fagan et al., 1994), and it phosphorylates most of the sites that are phosphorylated in vivo when Rb is phosphorylated in late G l (Matsushime et al., 1994). The recently discovered CDKIs may potentially act as tumor suppressers. Among them, the p 16 gene in particular, has been shown definitely to correlate with cancer. P16 is inactivated in a large number of cell lines derived from many different cancers. In some cases, the deletion frequency of the p 16 encoed gene exceeds 75% of tomour cell lines tested (Kamb et al., 1994; Nobori, et al, 1994). Another CDKI , p21, has been shown to be directly induced by p53, a central tumor suppressor, suggesting that p21 is a potential mediator of p53-dependent tumor suppression (El-Deiry et al, 1993; L i et al, 1994). p53 itself is the best-characterized cell cycle control protein linked to genetic mstabihty, which is the major cause of tumorigenesis (reviewed by Perry and Levine, 1993). In addition, C D K kinases have been found to phosphorylate in vitro many proto-oncogene products such as c-Src, c-Fos/Jun, c-Myb, c-Mos, suggesting the pos-sible direct connection between cancer and cell cycle control (Nigg, 1993). As our understanding of how cell cycle events are regulated advances, it is predicted that new 15 pharmacologic agents (for example, inhibitors of certain C D K kinases) are likely to be developed for cancer therapy. E. Paramecium and Its Cell Cycle Like all ciliates, each Paramecium tetraurelia cell has two types of functionally distinct nuclei in the same cytoplasm: two transcriptionally silent diploid germ line-micronuclei, and one transcriptionally active poly-genomic somatic macronucleus. The micronuclei only serve to transmit genetic information from one sexual generation to the next through the processes of conjugation or autogamy (self fertilization). During the latter part of micronuclear meiosis, the macronucleus breaks down into fragments which remain transcriptionally active, but are diluted and degraded with successive fis-sions. Micronuclei undergo meiosis to produce gametic nuclei that are reciprocally ex-changed (in conjugation), fuse and undergo two post-zygotic divisions, whose products differentiate into a new macronucleus and micronuclei. The differention of a new ma-cronucleus from a diploid germinal nucleus is accompanied by an extensive and repro-ducible reorganization of the genome, involving amplification to the final ploidy level (hundreds to thousands of copies), chromosome fragmentation (macronuclear chromo-somes range from 50 to 800 kb) and elimination of specific sequences. Paramecium cell cycle regulation has several unique features when compared to that of other "typical" eukaryotes (reviewed by Berger, 1988; Fig. 1-3). The two types of nuclei not only divide at different times, but undergo nuclear division by different mechanisms. Macronuclear D N A synthesis occupies most of the cell cycle, the amount of D N A is not exactly doubled before nuclear division. Initiation of macronuclear D N A synthesis occurs at 0.25 in the cell cycle and continues until the beginning of macronu-clear division at about 0.9 in the cell cycle (Rasmussen and Berger, 1982; Rasmussen et al., 1986). There is no discrete macronuclear G2 period. The macronucleus divides 16 IDS PCD Cell Division Mac G i s M Mic 0.25 0.5 0.75 1.0 |^- 90 min Fig. 1-3: Paramecium cell cycle control model. The control points (JDS and PCD) are shown with stages of macronuclear and micronuclear events. IDS is the intiation of macronuclear D N A synthesis and is located at 0.25 of the cell cycle. P C D is the main control point. At this point, cells become committed to cell division. The interval be-tween PCD and cell division is fixed (90 min). 17 passively by amitosis (no visible chromosomes, or spindles, or chromosome segrega-tion) and the resulting daughter cells frequently do not receive identical amounts of macronuclear D N A from their mother cell. Macronuclear D N A content is regulated through an incremental mechanism where all cells synthesize a certain amount of ma-cronuclear D N A regardless of their initial D N A content (Berger 1979). Micronuclear D N A replicates completely once before division as in typical eukaryotic nuclei and the period of D N A synthesis lasts for a much shorter time, from 0.47 to 0.69 in the cell cycle (Pasternak 1967). There is essentially no micronuclear G2 phase either. Micro-nuclear mitosis begins right after D N A replication and is completed about 20 minutes prior to cell separation (Adl and Berger, 1992). The two critical cell cycle transition points occuring in other eukaryotes, G l / S and G2/M transition, occur mParamecium, but are controlled from a single point in the Paramecium cell cycle. This point occurs at about 0.75-0.8 in the cell cycle (90 minutes prior to fission) (reviewed by Berger, 1988; Adl and Berger, 1992), where cells become irreversibly committed to division. At this time, the nature of the next cell cycle (meiotic or vegetative) is programmed (Berger, 1986), and the timing of initiation of D N A synthesis (IDS) i f the following cell cycle is to be a vegetative cell cycle is set (Berger and Ching, 1988). It is not clear what cell cycle regulators are involved in Paramecium cell cycle controL A temperature-sensitive cell cycle mutant in Paramecium, called ccl, has been extensively studied at both genetic and physiological levels (Rasmussen and Berger, 1986; Adl and Berger, 1992; Ad l and Berger, 1994). The ccl gene product is required for the execution of most of the major determinative events in the Paramecium cell cycle, including commitment to division, commitment to meiosis (autogamy) in the next cell cycle and duration of G l intervals (Adl and Berger, 1992; Berger, 1986; Berger and Ching, 1988). Therefore, the ccl gene product appears to be a regulatory protein which controls Paramecium cell cycle progression. Since it blocks cell cycle progres-sion at all the points in the cell cycle prior to commitment to division upon shift to the 18 restrictive temperature as well as macronuclear D N A synthesis, it is unlikely that ccl encodes a cdc2/cdc28 kinase. F. Organization of the Thesis The cell cycle regulatory mechanisms involving C D K kinases show a high de-gree of evolutionary conservation in both structure and function in higher eukaryotes. As mentioned above, it would be interesting to explore what drives the special features of the Paramecium cell cycle at the molecular level. Extensive previous work has been done on the physiology and genetics of the Paramecium cell cycle. This thesis initiates molecular analysis of Paramecium cell cycle control. The experimental results will be described in four chapters. In Chapter One, I will describe the development of an efficient synchronization method by centrifugal elutriation for Paramecium cells. This is the only efficient mass synchronization tech-nique for Paramecium and is essential for molecular analysis of the Paramecium cell cycle. Chapter Two details the isolation of a cdc2-Vke gene from Paramecium by a combination of various PCR (polymerization chain reaction) techniques and the com-parison with CDC2 sequences from other organisms. Paramecium CDC2PTA contains all the catalytic sites known to be required for kinase activity. About 83% of a second very closely related sequence was also identified and compared. In Chapter Three, the gene expression of the isolated Paramecium cdc2PtA during the cell cycle and in the stationary phase is presented. Based on the isolated se-quence, two different approaches were used to produce polyclonal antibodies. The Paramecium CDC2PTA antibodies were used to determine the protein level in the sta-tionary phase and exponential growth phase, as well as during the cell jcycle. Indirect 19 immunofluorescence was also performed to determine the subcellular localization of Paramecium CDC2PTA protein. In the last chapter, the gene product of Paramecium cdc2PtA was further stud-ied. Its ability to function as as a protein kinase was addressed, including analysis of phosphotransferase activity over the course of the cell cycle and complex formation with other proteins. Yeast pi3 s u c l Sepharose beads were incubated with Paramecium protein extracts and immunoblotted with Paramecium CDC2PTA antibody to see whether Paramecium CDC2PTA could be precipitated by this high affinity reagent for other p 3 4cdc2 proteins. Glycerol density gradient centrifugation and gel filtration were used to determine if Paramecium CDC2PTA forms complexes with any other protein. The fractionated proteins were immunoprecipitated with CDC2PTA antibody or pre-cipitated with plS*1"77, and assayed for histone H I phosphotransferase activity. 20 M A T E R I A L S A N D M E T H O D S A . Materials A l l restriction endonucleases and modifying enzymes were purchased from GIBCO B R L (Burlington, Ont., Canada) and Boehringer Mannheim (Germany) unless stated otherwise. A l l other chemical reagents were from either ICN Biochemicals (Costa Mesa, USA), SIGMA Chemical Co. (St. Louis, Mo., USA), or FISHER Scien-tific Co (New Jersey, USA) unless otherwise specified in the text. X-ray films were from Kodak (Canada). B. Stocks and Cell Culture Paramecium tetraurelia stock 51-S was grown at 27°C in phosphate-buffered Cerophyl medium, inoculated with Klebsiella pneumoniae (food organism) one day before use (Sonneborn, 1970). For hand-selection, small samples of cells were grown in Petri dishes. Mass cultures were obtained by growing up a single isolation line and feeding Paramecium cells twice a day by addition of one volume of the freshly inocu-lated medium To prepare mass cultures for centrifugal ehitriation, 2-3xl0 6 cells in a 2.8 liter flask 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 hr before ehitria-tion, these cells were diluted to a cell density of 500-800 cells/ml with fresh medium and refed every 12 hr. These mass cultures were grown in plastic boxes with 1.5-2 liter culture in each box. During this time, cells underwent roughly 3-4 divisions. The cell densities were monitored to ensure that the number of cells per ml did not exceed 2000, otherwise cells began to starve and cell cycle duration was changed. The population density was estimated by counting cells manually as described (Adl and Berger, 1991). For each ehitriation, about 1.5-2xl07 cells were used. 21 C. Centrifugal Elutriation Centrifugal elutriation was carried out with a Beckman JE.6-B centrifuge, JE.5 rotor and 30 ml chamber. A mass culture of about 1.5-2xl07 cells were concentrated by brief centrifugation (30 sec) at 1000 rpm and resuspended in 2.5 liter of freshly inocu-lated medium. Cells were loaded into the chamber of the elutriation rotor at 550 rpm with a flow rate of 85 ml/min at room temperature. After washing the cells in the chamber with 50 ml of fresh medium, flow rate was increased stepwise in 5 ml/min in-crements and fractions of 50 ml were collected at each flow rate beginning with the flow rate at which the first cells emerged from the chamber. For synchrony determina-tion, 35 fractions were collected. The rest of the cells were eluted by stopping the cen-trifuge rotor and were collected as "OFF" fraction. The whole elutriation procedure was completed between 1-1.5 hr depending on the initial volume. The fractionated cells were maintained at 27°C to determine synchrony and duration of the cell cycle. To collect elutriation synchronized samples for R N A and protein extraction, around 1.5-2xl07 Paramecium cells growing in -10 liters of the bacteria-inoculated Cerephyl medium were concentrated by brief centrifugation, resuspended in 2.5-2.8 liters of fresh medium and elutriated through a 30 ml chamber. The first 50 ml of efflu-ent containing cells was discarded. Subsequent fractions were pooled until the compos-ite contained about 10-12% of the total cell population. This synchronous population was maintained at 27°C and sampled periodically at one hour intervals starting one half hour after elutriation. Cells were harvested by centrifugation at 2500 R P M for 2 min, washed twice with cold Dryrs buffer (Sonneborn, 1970), and were immediately frozen using a -70°C methanol bath and stored at -70°C for R N A or protein extraction. To detennine the cell cycle duration and synchrony, a positive control of small synchronous samples was obtained by hand-selection of dividing cells from exponential growth-phase cultures. Usually, a set of 50 dividers was isolated into mdfvidual wells of 22 the glass depression slides over a period of 2-3 min. The same number of cells were randomly isolated from each of the elutriated fractions. The number of cells having reached division was noted at 30 min intervals. The kinetics of division of the synchro-nous cell population was analyzed by plotting the cumulative percentage of cells com-pleting division against time since fission or elutriation. D. Labeling of Paramecium with [3H]-Thymidine Escherichia coli, strain 15 Thy-, a mymidine-requiring auxotroph, was labeled with tritiated mymidine as described (Berger, 1984; Berger and Ching, 1988). Briefly, the bacteria were grown in M-9 medium supplemented with 4 p.g of thymine per m i Tritiated mymidine were added to a final concentration of 25 uCi per ml and the oilture was grown until the absorbance of the culture reached 0.4 at 695 nm The labeled bac-teria were concentrated by centrifixgation, washed twice with Cerophyl medium and stored at 4 °C prior to use. To label Paramecium cells, an aliquot (200 ul) of labeled bacteria was added to 1 ml of elutriation synchronized Paramecium cells and incubated for 20 min. The la-beled culture was washed twice with fresh Cerophyl medium and air-dried on an albu-min-coated slide. After fixation and staining with acriflavine, autoradiographs were prepared as described (Berger and Ching, 1988). The fraction of cells showing signifi-cant incorporation of labeled material into their macronuclei was plotted against time since elutriation synchronization. The median time of onset of D N A synthesis was de-termined by percentage of cells with labeled macronuclei 23 £ . DNA and RNA Isolation For genomic D N A isolation, Paramecium cells were harvested from exponen-tial phase cultures, pelleted, washed twice with DryPs buffer (Sonnebern, 1970), rysed at 65°C in 100 m M Tris (pH 8.0), 50 mM E D T A (pH 8.0), 200 m M NaCL 1% SDS and 1 mg/ml proteinase K for 3-6 hr till the lysates were clear. The lysis mixture was extracted three times with phenol:chloroformisoamyl alcohol (25:24:1), and twice more with chloroform only. D N A was purified by ethanol precipitation at -20° C for at least one hour. The D N A pellets were washed three times with -20°C 70% ethanol, vacuum dried and resuspended in TE buffer (10 m M Tris, 1 m M EDTA, p H 8.0). Total R N A was isolated from both exponentially growing and starved cells as described by Sambrook et al. (1989). Briefly, cell pellets were homogenized in 4 M guanidium thiocyanate, 0.1 M sodium acetate, 0.5 % sarcosyl. The homogenates were loaded onto a cushion of 5.7 M CsCl, 0.01 M E D T A (pH 7.5), centrifuged at 32,000 rpm for 22 hr at 20°C. R N A pellets were resuspended in diethyl pyrocarbonate (DEPC)-treated water and further purified by ethanol precipitation at -20°C. After washing three times with cold 70% ethanol in DEPC-treated water, the total R N A pel-lets were resuspended in 0.1% SDS prepared in DEPC-treated water. F. PCR Amplification The degenerate oligonucleotide primers for PCR amplification of a Paramecium cdc2 gene segment were designed to complement D N A sequences encoding the follow-ing conserved amino acid sequences of CDC2 kinase from various organisms: V A M K K I R (forward primer, 5 ' - C A T T C T A G A G T I G C I A T A A A / G A A A / G A T I A A A G -3', PAR-1), W Y R A P E (reverse primer, 5 ' -CATTCTAGAACT/CTCIGGIGCT/CCTA/ GTACCA-3 ' , PAR-3), and GCEFAEM(reverseprimer, 5 - C A T T C T A G A C A T T / C T C I GCA/GAAIATA/GCAICC-3 1 , PAR-4). The primers incorporated an Xba I site for 24 cloning of the PCR products. Total genomic D N A was used as template. The reaction contained 2 ug of genomic DNA, 100 pmol of each primer, 200 \iM of each dATP, dCTP, dGTP, dTTP, 2.5 U of Taq polymerase (BRL), 50 m M Tris-Cl (pH 8.0), 2.5 m M M g C l 2 , 0.05% Tween 20, 0.05% NP-40, in a final volume of 100 ul. After three cycles of denaturation at 95°C for 2 min, annealing at 48°C for 2 min, and extension at 72°C for 3 min, 30 additional cycles were carried out at 95°C for 30 sec, 50°C for 1 min and 72°C for 1 min. The conditions for all other PCR reactions were basically the same. Other minor changes will be specified elsewhere. G. Subcloning of PCR Products Amplification products of the predicted length were separated by gel electro-phoresis, excised from the agarose gel and purified with Sephaglas BandPrep Kit (Pharmacia), and digested with Xba I. The plasmid pBluescript II SK+/"(Stratagene) was used as cloning vector, digested with the same enzyme, and dephosphorylated by alka-line phosphatase (BRL). Both PCR D N A fragments and digested plasmid D N A were phenol-chloroform extracted, purified by ethanol precipitation at -20°C. After washing in 70% ethanol and resuspending in water, they were ligated together at 15°C for 16 hr using bacteriophage T4 D N A ligase. The resulting ligates were transformed into com-petent E. coli strain DHa5 as described by Sambrook et al. (1989). Briefly, the plasmid D N A was introduced into bacteria by heat-shock at 43°C for 90 sec. The infected bac-teria were grown in liquid L B medium (1% peptone, 0.5 % yeast extract, 1% NaCl), then plated on 1.5% agar in L B medium containing 30 ul of 100 m M IPTG and 110 ul of 20 mg/ml X-gaL and incubated overnight at 37°C. The recombinant transformants were identified by white-blue selection. Plasmid D N A with inserts was isolated by mini-preparation (Sambrook et al., 1989) and further confirmed by gel electrophoresis after Xba I digestion. The recombinant pGEX-2T plasmid D N A was introduced into the 25 same cell strain in the same way, except for that no IPTG or X-gal was included in the medium. H. Isolation and Sequencing of Genomic and cDNA cdc2 Gene The rapid amplification of cDNA ends (RACE) (Frohman 1990) was used to isolate the 3'-end of the gene. Total R N A was used for reverse transcription. Single-stranded cDNA was synthesized by primer extension reaction using an oligo-dT17 primer (5 ' -GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3 ' , dT-1) orhex-anucleotide random primers. The extension reaction was carried out in 50 m M Tris-HC1 (pH 8.4), 50 m M KC1, 8 m M MgCl 2 , 2.5 m M DTT, 0.8 m M of each dATP, dCTP, dTTP, dGTP, 1 fig of total RNA, 50 ng of primers, and 200 U of M - M L V reverse transcriptase (BRL). The PCR reaction was carried out using the cDNA as template, the adapter primer for oligo-dT17 ( 5 ' - G A C T C G A G T C G A C A T C G A - 3 ' , ADA-1) and another specific oligonucleotide ( 5 ' - G G T C T A G A G A G A T C T C T C T T C T C A A A GAG-3', PAR-6) based on the Paramecium cdc2 fragment obtained by PCR (Fig. 2-1). The R A C E technique was also tried to obtain the 5'end sequence. After the synthesis of the first strand cDNA, a poly-A tail was added to the 5'end of the gene by terminal transferase. The dT-1 primer and an antisense primer designed from the PCR fragment (5 ' -CGTCTAGACTCTGATGGCAGTACTAGG-3 ' , PAR-5) were used for P C R It was not successful for obtaining the 5'-end sequences by R A C E . Another approach, the rapid amplification of genomic ends (RAGE) developed by Mizobuchi and Frohman (1993), was used to isolate the 5'-end region. Briefly, Paramecium genomic D N A was digested with Hind U l and ligated into Hind Hi-digested plasmid DNA. The ligates were used as templates for PCR using one primer (5 ' -AAGTTGGGTAACGCCAG-3 ' , BS-1) from pBluscript sequence, and an antisense primer (5 ' -GGCTCGAGGCCTTT CTCTTTGGGGAATTG-3 ' , Fig. 2-1, PAR-8) from the Paramecium cdc2 fragment 26 obtained by PCR with degenerate primers. This PCR product was used as a template for a second PCR using two internal primers, one from the pBhiescript ( 5 - C C A G T C A CGACGTTGTA-3 ' , BS-2) and another from the Paramecium cdc2 PCR fragment (5'-C G A G C T T C T T A T T T G A A T G C A C A A C - 3 ' , Fig. 2-1, PAR-10). The reamplified PCR products were separated by electrophoresis, excised from the agarose gel and purified as described above. The PCR fragments were filled-in by the large fragment of Klenow D N A polymerase (BRL), were then ligated into Sma I-digested pBluescipt at 25°C for 16 hr in the presence of Sma I to prevent plasmid self ligation, and transformed into DHcc5 cells as described above. Plasmid D N A was prepared from the white colonies. The inserts were confirmed by both restriction enzyme digestion and sequencing by a chain termination method using primer PAR-10. A l l D N A sequencing was performed by the dideoxy cham-termination method using double-stranded recombinant plasmid as T7 templates (Sanger et al., 1977). A Sequencing Kit (Pharmacia) was used for all se-quence determinations, following the included protocol, except that double the sug-gested amount of template D N A was used and denaturation reactions were carried out in 20 ul instead of 10 p i The whole cDNA and genomic D N A sequences were isolated by PCR using a forward primer (PAR-11, 5 ' - G C G G A T C C A T G A A A T C T A A T A A A C T G G - 3 ' ) from the 5'-end, and a reverse primer (PAR-14, 5 - G A A A G G T T T T A T G A A A T T T G A T A T ATA-3') from the 3'-end after the stop codon. The PCR products were cloned into plasmid D N A and the whole gene was sequenced from both strands by using different oligonucleotides designed from the Paramecium cdc2PtA sequence. I. D N A Probe Preparation The probe was labeled by digoxigenin-11-dUTP (DIG-11-dUTP, Boehringer Mannheim) with PCR, using primers PAR-11 and PAR-12 (5 ' -GCGAATTCATAGA 27 G G A A A C T C T T C A C - 3'). Total R N A was used for cDNA synthesis by random prim-ing and resulting products were used in the intial PCR reaction using the above two primers. The expected fragment was purified from the agarose gel and 1 ng was used for the labeling PCR reaction. This PCR reaction was carried out as described before except that 133 u M DIG-11-dUTP was included and only 67 u M dTTP was used in the reaction. The incorporation of DIG-11-dUTP into PCR products was monitored by gel electrophoresis, since D N A fragments containing DIG-11-dUTP migrated more slowly on an agarose gel. The labeled PCR products were purified by ethanol precipita-tion and added to the hybridization solution. Usually, around 50 ng of DIG-11-dUTP labeled PCR product was obtained from a 100 ul reaction and 10 ng were used in 15-ml hybridization solution for Southern and Northern blotting analysis. J . Southern Blot Analysis For Southern blots, 20 ug of genomic D N A were digested with 7 different re-striction enzymes (Fig 2-5), separated on an 0.8% agarose gel and transferred to Hy-bond-N+ membrane (Amersham) for 2.5 hr using the downward technique in 0.4 M NaOH as described by Koestsier et al. (1993). Hybridization and detection were per-formed as described by Engler-Blum et al. (1993) using a DIG Luminescent Detection Kit from Boehringer Mannheim Briefly, after transfer, the membrane was neutralized in 2xSSC for 10 min and dried at 80°C for 15 min. Then the membrane was incubated with prehybridization solution (0.25 M N a 2 H P 0 4 (pH 7.2), 8% SDS, 1 m M E D T A , 0.5% blocking reagent (Boehinger Mannheim)) at 67°C for at least one hour. The membrane was further incubated with 15 ml of prehybridization solution containing 10 ng of DIG-11-dUTP labeled Paramecium cdc2PtA D N A probe at the same tempera-ture for 8 hr to overnight. The filter was washed with pre-warmed washing buffer (20 mM Na 2 HP0 4 , 1 m M E D T A and 1% SDS) at 64°C three times for 20,min each. For 28 detection, the filter was washed briefly in detection washing buffer (0.1 M maleic acid, 3 M NaCL 0.3 % Tween-20 (pH 8)), blocked in detection buffer (detection washing buffer containing 0.5 % blocking reagent) for 45 min at room temperature with shak-ing, incubated for one hr with anti-DIG antibody conjugated to alkaline phosphatase (diluted 15000 times in detection buffer). After three washes (15 min each) in detection washing buffer, the filter was transferred to developing buffer (100 m M Tris-HCl, 100 m M NaCL 50 m M MgCl 2 , p H 9.5) containing Lumigen-PPD (4-methyl-4(3-phosphatephenyl)-spriro-(l,2-dioxetane-3,2'-adamantane) for 5 min as described by the supplier. The filter was wrapped in a plastic bag, preincubated at 37°C for 30 min and exposed to X-ray film. K . Northern Blot Analysis Ten ug total R N A was separated on a 1.2% agarose/formaldehyde gel as de-scribed by Sambrook et al. (1989), except that 0.22 M formaldehyde was used for both gel and electrophoresis running buffer. R N A was blotted to Hybond-N4" membrane by downward alkaline capillary transfer as described by Chomczynski (1992), except that two buffer containers were used for the transfer setup instead of one. Briefly, after gel electrophoresis, R N A was downward transferred to the membrane using alkaline transfer buffer (3 M NaCL 8 m M NaOH and 2 mM sarkosyl) for 1.5 hr. The membrane was neutralized in 0.2 M phosphate buffer (pH 6.8) for 10 min, dried at 80°C for 15 min, incubated in Northern prehybridization buffer (0.25 M Na2HP0 4 (pH 7.2), 1 m M EDTA, 7% SDS, 1% blocking reagent (Boehringer Mannheim), 50% Formamide) at 45°C for at least one hr, and hybridized at 45°C for 16 hr using a D I G - l l - d U T P la-beled probe (about 1 ng per ml) in Northern prehybridization buffer. Washing and de-tection steps were performed exactly as for the Southern blotting described above. The signals of the chemihimin escence was quantified by using a Molecular Dynamics densi-tometer (USA). L. Determining Relative Level of cdc2PtA Transcripts by RT-PCR Fifty dividers were hand-selected at one hour intervals for 10 hrs. After more than 90% of the first 100 cells had divided, all samples were harvested by brief cen-trifugation and washed once with Dryl's buffer. Total R N A was isolated from these samples by the procedure of Xie and Rothblum (1991). Briefly, cells were lysed by vortexing in 200 ul of lysis buffer (4 M guanidium thiocyanate, 0.5 % sarkosyL 25 m M sodium citrate (pH 7), 0.1 M B-mercaptoethanol) for 30 sec. One tenth volume of 2 M sodium acetate (pH 4), 1 volume of water-saturated phenol (pH 4 or less) and 1/10 volume of chloroform/isoamyl alcohol (49:1) were added to the h/sates. After vigorous vortexing and 5 min centrifugation in a microcentrifuge , R N A retained in the top aqueous phase was precipitated at -20°C with isopropanal and washed with cold 75 % ethanol (-20°C) prepared in DEPC-treated water. The final R N A pellets were resus-pended in 10 pi of 0.1 % SDS in DEPC-treated water. Reverse transcription was car-ried out in PCR reaction buffer by random priming and the resulting cDNA products were used directly for PCR amplification. Primers PAR-11 and PAR-12 were used for PCR reaction and encompassed the two introns. The PCR reaction was carried out for only 25 cycles in the presence of DIG-11-dUTP as described for D N A probe prepara-tion before. The resulting PCR products were separated on 1 % agarose gel, trans-ferred to Hybond-N4" membrane and directly detected by incubating with anti-DIG al-kaline phosphatase conjugated probes (Boehringer Mannheim) and chemiluminescence as described for Southern blotting. The linearity between R N A imput and the yields of the PCR products was also quantified by a Molecular Dynamics densitometer. 30 M . Preparation of Paramecium Protein Extracts Four volumes of protein lysis buffer (50 mM Hepes, 25 mM IJ-glycerolphosphate, 10 mM NaF, 150 mM NaCl, 0.5 mM Na3VC«4, 5 m M benzamidine, 15 m M E G T A , 5 mM EDTA, 1 m M DTT (dithiothreitol) and 0.1% Triton X-100, p H 7.4) were added to cell pellets containing the following cocktail of proteinase inhibitors: phenylmethyl sulfonyl floride (PMSF), 50 ug/ml; leupeptin, 2 ug/ml; aprotinin, 4 ug/ml; tosyl phenylalanine chloromethyl ketone (TPCK), 10 ug/mL pepstatin; 5 ug/ml. After brief vortexing, cells were sonicated for 4x15 sec on ice. Cell extracts were centrifuged at 60,000 rpm for 30 min at 4°C. The supernatants were frozen immediately at -70°C. Tetrahymena proteins were prepared the same way. For synchronized small samples, four volumes of the hypotonic protein lysis buffer (the same buffer as described above except that only 10 mM NaCl was included in the buffer) were added to the cell pellets, and vortexed for 3x20 sec. NaCl was then added to 150 m M to cell lysates and the lysates were kept on ice for 10 min. Cell extracts were centrifuged at full speed for 10 min at 4°C in a microcentrifuge and the supernatants were centrifuged for the second time for 5 min. The resulting super-natants were frozen at -70°C. Aliquots were taken for protein quantitation using the Bradford method (Bradford, 1976) using 1 Hg/ul of B S A (bovine serum albumin) as a standard. N. Expression in E. coli and GST-Fusion Protein Purification Due to different codon usage in Paramecium, the whole gene for cdc2PtA could not be expressed in bacteria. Paramecium uses only one stop codon (TGA) and the isolated Paramecium cdc2PtA contains 6 internal T A A (glutamine) codons that are 31 used as stop codon in other organisms (Fig. 2-1). However, the first third of the se-quence from the N-terminus contained no T A A codons and was chosen for partial ex-pression in E. coli. Total R N A was isolated from Paramecium cells and reverse tran-scribed by random priming as described before. The resulting cDNA was used as tem-plate for PCR by primers PAR-11 and PAR-12, which were located at the beginning of the gene and just before the first T A A codon, respectively (Fig. 2-1). A 350 bp frag-ment was obtained as expected, purified and digested with EcoR I and BamH I. The resulting PCR fragments were subcloned into EcoR VBamH I-digested pGEX-2T plasmid (Pharmacia) and transformed into DHa5 bacteria. The junction of the recombi-nant pGEX-2T and the right order of the insert sequence were confirmed by D N A se-quencing. Production of the recombinant glutathione S-transferase (GST)-CDC2PTA fusion protein was induced by 0.2 mM isopropyl-D-thiogalactopyranoside (JJPTG). Protein extraction and purification were carried out as described by Smith and Johnson (1988). Briefh/j the bacteria pellets were lysed by sonication in 10 volumes of extrac-tion buffer (PBS containing 2 m M E D T A (pH 8), 0.1% mercaptoethanoL 5 m M ben-zamidine and 0.2 m M PMSF). Upon lysis, Triton X-100 was added to 1% and the lysates were centrifuged at full speed on a microcentrrfuge at 4°C for 10 min. The su-pernatant was mixed with 1/10 volume of glutathione-conjugated agarose beads (Sigma) with rotation at 4°C for 30 min. After three washes with the extraction buffer containing 1% Triton X-100, the GST-CDC2PTA fusion protein was eluted by freshly prepared 10 m M glutathine in 50 mM Tris (pH 8.0) (final p H 7.5). The purity was monitored by SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and Coomassie blue staining. The identification of the fusion protein was further confirmed by im-munoblotting with anti-PSTAIRE antibody using GST protein as a control. The anti-PSTAIRE antibody cross reacted only with GST-CDC2PTA fusion protein and did not react with GST protein alone. 32 O. Production and Purification of Antibodies Both GST-CDC2PTA fusion protein and synthetic peptides were used for anti-body production. A 15 amino acid peptide was chosen from the C-terminal region of the Paramecium cdc2PtA sequence ( C K E L P E Q V K K L Y V N V K ) with an additional cysteine at the amino end. The antibody production and purification were carried out as described by Sanghera et al. (1992). Briefly, New Zealand white rabbits were used to produce antibodies against Paramecium cdc2PtA. Intramuscular injections were carried out subcutaneously every 4 weeks with K L H (keyhole limpet hemocyanin)-coupled peptides or GST-CDC2PTA fusion protein. Both peptide and GST-CDC2PTA fusion protein were emulsified in Freund's adjuvant. Complete adjuvant was used for the first injection, and incomplete adjuvant for all subsequent injections. The first injection used 250 ug of coupled peptide and 500 ug of GST-CDC2PTA fusion proteins, and re-spectively, 100 ug of coupled peptide and 200 ug of GST-CDC2PTA fusion protein for the subsequent boosting. The whole body blood was collected 10 days after the fifth injection. To purify the antibodies, the sera from these animals was applied to an agarose column to which the immunogen peptide or GST-CDC2PTA fusion protein were thio-linked. The serum was passed through the column 4-6 times. After extensive washing with PB S, antibody was eluted from the column with 0.1 M glycine, p H 2.5. Subse-quently, the antibody solution was neutralized to pH 7.0 with saturated Tris. The anti-PSTATRE polyclonal antibody was a gift from Dr. Pelech (Kinetek Biotechnology Corp. and Faculty of Medicine, UBC). The synthetic peptide sequence was: EGVPSTAIREISLLKE. 33 P. Protein Electrophoresis and Immunoblotting Proteins were resolved on 12-15 % SDS-polyacrylamide gels as described by Laemmli (1970) and Harlow and Lane (1988), transferred to Immobilon-P membrane (Millipore), and immunoblotted with the above antibodies as described (Harlow and Lane, 1988; Sheng and Schuster, 1992). Briefly, gel electrophoresis and protein trans-fer were performed using the apparatus purchased from Hoeffer. After transfer, the membrane was rinsed in TBS (150 mM NaCL 10 mM Tris, pH 7.4), incubated with Western blocking buffer (1 % non-fat milk in PBS) at 37°C for 3 hr with shaking, washed for 15 min with 0.1 % B S A in TBST (TBS containing 0.5 % Tween-20 (v/v)) (Western washing buffer). The filter was then incubated with the primary antibody di-luted in antibody solution (TBST containing 1 % BSA) for 2 hr, washed three times with the Western washing buffer and blocked with 1 % milk in PBS for 30 min. After three washes in TBST, the membrane was incubated with the alkaline phosphatase conjugated goat anti-rabbit antibodies from Bio-Rad (diluted 1:3000 in antibody solu-tion). The colorimetric approach using BCD? (5-Bromo-4 chloro 3-indolyly phosphate) and N B T (p-nitro blue tetrazolium) as substrates was used for visualization as specified by the supplier (Bio-Rad). Q. Immunofluorescence Sa\mple preparation for immunofluorescence analysis was performed in a mi-crocentrifuge tube. Paramecium cells were permeahilized for 5 min in P H E M buffer (60 m M PIPES, 25 mM HEPES, 5 mM EDTA, 2 mM MgCl2 ) containing 1 % Triton X-100, 200 u M PMSF, 2 ug/rnl leupeptin and 4 pg/ml aprotinin. Cells were fixed in freshly prepared 2.5 % paraformaldehyde for 10-15 min, or in methanol at -20°C for 15 min. Following 3 washes in PBS containing 1 % BSA, 0.1 % Tween-20, 5 m M E D T A and 2 m M MgCl2, cells were incubated with affinity purified anti GST 'CDC2PTA anti-34 bodies at a dilution of 1:100 in PBS containing 3 % BSA for 1 hr. After 3 washes, cells were resuspended in a 1:200 dilution of FITC-conjugated goat anti-rabbit immuno-glubulin antibody (BRL) in 3 % BSA in PBS for 45 min. To stain the nuclei, 0.5 ug/ml of DAPI was added to the secondary antibody incubation and incubated for another 5 min. Cells were mounted on a slide in 50 % glycerol in PBS after 3 washes as above and another 3 washes in PBS. Cells were viewed using a Zeiss Axioscope and pictures were taken right away. R. Metabolic Labeling of Paramecium Cells Paramecium cells were cultured at 27°C with Cerophyl medium and concen-trated to around 5xl03 per ml in freshly inoculated medium. Concentrated cells were 35 35 incubated with [ S]methionine/[ S]cysteine (ICN) at 50 uCi/ml for 2 hr with occa-sional mixing by pipetting up and down, and harvested by brief centrifugation. After washing twice with cold Dryl's buffer, cells were frozen at -70°C or lysed directly in hypotonic lysis buffer as described before. S. S. pombe Cells and Protein Extraction Schizosaccharomyces pombe strain NRCC 2744 (Y. N. Lee, McGill) was grown at 30°C with shaking in Y E medium (0.5% yeast extract, 3% glucose and 30 (j,g/ml leucine). Protein extraction was carried out as described by Daya-Makin et al. (1992). Briefly, harvested cells were washed twice with ice-cold PBS and resuspended in ice-cold lysis buffer with all of the proteinase inhibitors described for preparing Paramecium protein extracts, one volume of glass beads was added and vortexed for 4 times of 30 sec each time for a total period of 2 min, and protein extracts were col-lected by centrifugation at full speed at 4°C for 5 min in a microcentrifuge. The super-35 natants were recentrifuged at 60,000 rpm for another 20 min at 4°C, and the resulting supernatants were assayed for protein concentration and stored at -70°C. T. pl3s"c' Precipitation S. pombe pl3sucl was expressed in E. coli, purified, and linked to cyanogen bromide (CNBr)-activated Sepharose CL4B (Brizuela et al, 1987). The final prepara-tion of beads contains approximately 3.5 mg/ml of Tpl3suc'. Crude protein extracts or fractionated proteins after density gradient sedimentation or gel filtration, were diluted to around 200 ng per ml with cold bead buffer (50 m M Tris (pH 7.4), 250 m M NaCL 5 mM NaF, 5 m M EDTA, 5 m M EGTA, 0.1% NP-40, and 5 u M benzamidine) contain-ing the cocktail of proteinase inhibitors described above (see page 30). The protein fractions were incubated then with pl3* u c / beads (about 100 pi of beads per mg pro-tein) at 4°C with rotating for 2-4 hr. The beads were washed five times with cold bead buffer and bound material was used for histone HI kinase activity assay or eluted with LaemmU's SDS sample buffer for analysis on Western blots. U. Immunoprecipitation One standard immunoprecipitation reaction consisted of 5 pi of polyclonal se-rum or 5 pg of affinity purified antibody with 200 pg-1 mg of Paramecium protein extracts in a total volume of 500 pi of IP buffer (50 m M HEPES (pH 7.4), 250 m M NaCl and 0.5 % Triton X-100) with all proteinase inhibitors used in the preparation of protein extracts. For competitive immunoprecipitation, purified antibody or anti-serum was incubated with 2 ug of synthetic peptide or 10 ug of GST-CDC2PTA fusion pro-tein in 50 pi of IP buffer for 30 min at 4°C. Then protein extracts were added and the reaction volume was brought up to 500 pi with IP buffer. After incubation for 2 hr on ice, 20 pi of a 1:1 slurry of protein A-agarose (BRL) were added to each sample. The 36 mixture was rotated at 4°C for another hour and the pellets were washed 3-5 times with IP buffer. For immune complexes destined for immunoblotting or autoradiograghy, 50 ul of Laemmli buffer was added to the final pellets. These samples were heated to 100°C for 3 min prior to SDS-PAGE. For those samples which were used for kinase activity assay, the pellets were washed twice more with 50 mM HEPES (pH 7.4) and 1 mM DTT, twice more in kinase assay buffer, and used for kinase activity assay as de-scribed below. V. Glycerol Density Gradient Centrifugation For sedimentation analysis, protein extracts (approximately 5 mg in 300 ul) were loaded on 9 ml 10-30 % glycerol gradients prepared in protein lysis buffer con-taining the proteinase inhibitors as described before. The gradients were sedimentated at 40,000 rpm at 4°C for 36 hr in an SW41Ti rotor (Beckman) as described (Zhang et al, 1994). The fractions (about 260 ul each) were collected by drops from the bottom by puncture of the centrifuge tube. Odd numbered fractions of Paramecium protein extracts were mixed with Laemmli's sample buffer and boiled for 3 min for Western blotting analysis. Since the anti-PSTAIRE antibody did not work very well on S. pombe crude extracts, fractionated yeast extracts were precipitated with pl3*uc/ and used for Western blotting analysis. Even numbered fractions were assayed for histone HI kinase activity. For each assay, about 65 ul of the fractionated proteins were either precipi-tated with pl3sucl beads or immunoprecipitated with 5 ul of Paramecium CDC2PTA antiserum After extensive washing, the recovered kinase on either $l3sucl beads or protein-A beads was assayed for histone H i phosphorylation by scintillation counting as described later. The mean of three independent experiments was plotted against the fraction number. For [35S]-methionine labeled samples, 10 mg of labeled protein ex-tracts were applied on the glycerol gradient and the fractionated proteins were im-37 munoprecipitated with Paramecium CDC2PTA antiserum. The precipitated proteins were separated on SDS-PAGE, transferred to the membrane, dried and detected by autoradiography. W. Gel Filtration A Superose 12 (Pharmacia) gel filtration column was equilibrated with lysis buffer and calibrated with alcohol dehydrogenase (150 kDa), ovalbumin (45 kDa) and carbonic anhydrogenase (29 kDa). About 5-10 mg of protein extract in 200 pi of lysis buffer were loaded onto the column and eluted with lysis buffer at a flow rate of 0.4 rnVmin. Fractions of 250 pi were collected and 4 ul were used for assay of crude his-tone HI kinase activity: Alternatively, 125 pi from each fraction were mixed with 30 ul of pl3*uW beads, and the pellets were tested for histone HI kinase activity. To detect the presence of CDC2-like proteins, 100 ul from each fraction were boiled in IX SDS-PAGE sample buffer and immunoblotted with anti-PSTAIRE antibody. X. Kinase Activity Assay Kinase assay cocktail contained 50 nM cAMP-dependent protein kinase inhibi-tor peptide (Sigma), 0.5 mg/ml bovine histone HI or 1 mg/ml casein purified from bovine milk (Sigma) or 1 mg/ml GST-Rb fusion protein (gift from Dr. Ewen, Dana-Farber Cancer Institute, Boston), 5 mM EGTA, 15 mM MgC^, 50 mM Tris (pH7.5), in a final volume of 25 pi. This cocktail was added to immunoprecipitated protein-A beads or pi3sucI-precipitated beads. Kinase reactions were started by the addition of radioactive [y-32P]-ATP to 50 pM (2000 cpm/pmol, about 2.5 pCi per reaction) and were incubated for 20 min at 30°C. For most of the kinase activity assays, the reaction was terminated by spotting 15 pi aliquots on to 2.5x3 cm pieces of Whatman p81 phosphocellulose paper. After 30 seconds, the filters were washed at least 5 times in a 38 solution of 0.1% phosphoric acid for a total length of at least 30 min. The wet filters were transferred to 7-ml plastic scintillation vials, scintillation fluid (3ml) was added, and the samples were counted in a scintillation counter to determine the incorporation of [32P] into the basic substrate histone HI. Each datum was the mean of three inde-pendent assays. Alternatively, the kinase assay reaction was terminated by adding SDS-PAGE sample buffer and boiled for 3 min. The phosphorylated substrate proteins were separated by SDS-PAGE and phosphorylated proteins were determined by autora-diography. 39 CHAPTER ONE SYNCHRONIZATION OF PARAMECIUM CELLS BY CENTRIFUGAL ELUTRIATION 1.1 INTRODUCTION It is difficult to determine precisely the relative timing of the various events that comprise the cell cycle in eukaryotes, especially when attempts are made to correlate events that occur on a cellular scale (e.g., the commitment point to cell division) with those that occur on a molecular scale (e.g., CDK/cyclin kinase complex formation), because most molecular events cannot be studied in single cell assays. The relationship between cellular and molecular events can be studied by using cell synchronization to produce a large number of cells that occupy a narrow window within the cell cycle. Synchrony can be achieved by selection or induction methods. Cell growth oc-curs at a constant rate which is reflected in a smooth increase in volume as the cells progress through the cell cycle. Centrifugal elutriation separates cells on the basis of seclimentation properties with minimum perturbation of metabolic function. An essen-tially homogeneous population of synchronous cells can be obtained by selecting the smallest, newly divided cells. This method has been used to synchronize a wide variety of eukaryotic cells from yeast to human cultures. Synchrony can also be induced. For example, in yeast, when appropriate tem-perature-sensitive cells are shifted to a restrictive temperature for a certain time, prog-ress through the cell cycle will be blocked at the cell cycle arrest point of that particular mutant. After several hours at the restrictive temperature to ensure that all cells have reached the block point, the cells can be shifted back to the permissive temperature at which time all the cells recover and continue through the cell cycle, synchronized from 40 the arrest point. Chemical treatments can also induce synchrony. DNA synthesis inhibi-tors (e.g., hydroxyurea) block cells in S phase. Microtubule destabilizing agents (e.g., nocodazole) arrest cells at metaphase. In S. cerevisiae, the purified peptide mating pheromone alpha factor can also be used to synchronize cells at "START" in G l phase. When washed free of pheromone, all the yeast cells return to the cell cycle from the same point (Gl). Paramecium cells are big (120-140 umlong) compared to most eukaryotic cells, and swim vigorously. The only reliable technique for obtaining synchronous populations has been manual selection of dividers (Sonneborri, 1970). This technique is only useful for small samples and impractical for the collection of sufficient quantities of synchronous cells for biochemical analysis. Previous attempts to obtain a synchronous mass cultures of Paramecium have met limited success (Aufderheide, 1976). Develop-ing a synchronizing method was an essential step to initiate molecular analysis of Paramecium cell cycle regulation. In this chapter, a centrifugal elutriation procedure to obtain a synchronized mass Paramecium cell culture is described. 1.2 RESULTS 7 The 30 ml chamber of the ehitriator can hold up to 2.5x10 Paramecium cells. I 7 routinely used 1.5-2x10 cells. All the cells were loaded into the chamber at 550 rpm with a flow rate of 85 ml/min and then eluted by a stepwise increase of flow rate. Frac-tions of 50 ml were collected and 50 cells from each fraction were grown in a depres-sion slide. The number of divided cells was counted at half hour intervals and the cumu-lative percentage of dividers was plotted against time after elutriation. As is shown in Figure 1-1, the cell cycle length from the early fractions was similar to that of the small population of hand-selected control cells (fractions 3-15). However, the smallest cells 41 from the very earliest fraction (fraction 1, Fig. 1-1) did not grow well and about 30% of them did not divide even after all cells from other samples had divided. It was assumed that this fraction contained a substantial numbers of cells that were physically damaged during the elutriation process. It is likely that some of those smallest cells were physi-cally separated by the elutriation forces before the two daughter cells were physiologi-cally ready to do so. A fraction containing about 2xl06 synchronous cells was obtained from the smallest 10-15% of an asynchronous cell population. The resulting synchrony of divi-sion was as good as that of a small population of hand-selected control cells (Fig. 1-1). Like the control samples, cells started dividing about 7 hr after the elutriation and more than 80% of the cell population finished division within 2 hr. The correlation between elutriation and hand-selected cells in the time elapsed between synchronization and the median time of division indicated that the elutriated cells were selected as newborn Gl stage cells. This is further supported by the DNA synthesis data using [3H]-myrnidine labeling (Fig. 4-4). These elutriation synchronized fractions (3-15, Fig. 1-1) represented about 12% of the original cell population (Fig. 1-2). Cells from later fractions were not well synchronized. Dividers were present as early as half an hour after elutriation and through all the subsequent time points (fractions 25 and 30, Fig. 1-1). No more than 50% of cells divided within a 2 hr period from these later fractions. This result indicates that although the cells from later frac-tions were all separated on the basis of their size, they were not located at the same point of the cell cycle. Therefore, in Paramecium, like yeast and mammalian cells, after early Gl , cell size alone is not a precise determinant of a cell's position within the cell cycle. I conclude from this study that only around the first 12% of cells after the cen-trifugal elutriation were well synchronized at early G l phase and cells from later frac-42 tions were not synchronized. The ehitriation selected G l cells progressed synchro-nously through the cell cycle and typically started dividing at 6.5-7.5 hr after elutriation like hand-selected cells. Cells of the very earliest fractions were physically damaged during elutriation and could not be used as synchronized early Gl cells. 120 0.5 2.5 4.5 6.5 8.5 10.5 12.5 Time (hr) Fig. 1-1. Synchronization of Paramecium cells by centrifugal elutriation. The cumula-tive percentage of cells having reached division is plotted as a function of time since elutriation fractionation. Fifty cells from each fraction were examined. Elutriation frac-tions 1, filled squares with crosses inside; 3, open triangles; 15, open circles; 25, open squares; 30, filled squares; hand-selected control sample, pluses. 1 43 O O o (/) "(D O (D E 16020 14020 -12020 -10020 -8020 -6020 -4020 2020 -\ 20 synchronized G1 population -r-f-1 13 17 Fraction 21 'OFF" 25 29 M00 k o a 0 Fig. 1-2. Fractionation of Paramecium cells by centrifugal elutriation. The cumulative percentage and number of elutriated cells are plotted as a function of elutriation frac-tions. The cumulative number of cells is shown on the left and cumulative percentage of cells elutriated on the right. "OFF" fraction contained cells collected after the centrifuge was turned off in which all cells were elutriated out of the chamber. Note that fractions 1-15 which were synchronized at G l phase contain around 12% of the whole popula-tion. ' -44 1.3 DISCUSSION The conservation of the central control system by CDC2 or its related kinases in eukaryotes occurs from fungi to green algae, plants and animals including humans. However, the lack of a method for obtaining large populations of synchronous cells has undermined analysis of Paramecium cell cycle regulation. In general, the only fre-quently used technique for obtaining synchronous populations of Paramecium has been manual selection of dividers developed by Sonneborn (1970). A few hundred dividers can be easily selected under the microscope in a few minutes by this method. However, the number of cells obtained is only enough for physiological and genetic analysis. Based on the density-labeling synchronization method developed for Tetrahymena by Wolfe (1973), Aufderheide (1976) reported a technique for mass selection of synchro-nous Paramecium cells. Paramecium cells cease feeding approximately 10 min before and after fission. By presenting granular tantalum to the population, the unlabelled sub-population (which are immediate pre- and post-fission cells) can be separated from the remainder of the population (which are the cells located at the rest of the cell cycle by the time of tantalum feeding) by gradient centrifugation. The selected subpopulation will be synchronous for subsequent cell division when resuspended in fresh medium But only a few thousand synchronous cells can be obtained by this method and there-fore this method is still impractical for biochemical analysis. Synchronization induced by DNA synthesis inhibitors and microtubule destabi-lizing reagents has been used frequently in mammalian systems and other eukaryotic cells. Paramecium has two distinctive nuclei The macronucleus and micronucleus enter the cell cycle at different times in the cell division cycle (Berger, 1988). Macronuclear DNA synthesis occupies most of the cell cycle and the macronucleus divides near the end of the cell cycle. Micronuclear DNA replication starts at the middle of the cell cy-cle, extends for a very short period of time and is completed before the end of macro-45 nuclear DNA synthesis. The micronucleus divides before the macronucleus. If Para-mecium cells are treated with drugs which affect DNA synthesis or microtubule polym-erization, the two nuclei will be blocked at different points. For example, any microtu-bule depolymerizing agents added to the cells at a time before macronuclear division and after micronuclear division will produce amacronucleate daughter cells. In fact, Caron and Ruiz (1992) used nocodazole to isolate micronuclear specific DNA se-quences from Paramecium. DNA synthesis inhibitors will affect DNA replication of the two types of nuclei similarly. So, it is impossible to synchronize Paramecium cells by using these drugs. Synchronization by cell cycle mutants does not work in Paramecium either, since none of the Paramecium cell cycle mutants isolated so far blocks cell cycle at a single regulatory point. I conclude from this chapter that the only practical way to syn-chronize Paramecium in mass culture, at this time, is the centrifugal elutriation method. By this method, l-2xl06 isolated early G l cells can be readily well synchronized. These cells were collected and allowed to go through the cell cycle synchronously, and used for the subsequent molecular and biochemical analysis in the following chapters. 46 CHAPTER TWO ISOLATION OF A HOMOLOGUE OF THE C E L L C Y C L E CONTROL GENE CDC2 FROM PARAMECIUM TETRAURELIA 2.1 INTRODUCTION One of the key components in the cell cycle regulatory cascade is the ser-ine/threonine specific protein kinase, p24cdc2/CDC28. This kinase was originally identified as the S. cerevisiae CDC28 gene product and later as the & pombe cdc2gene product. The protein products of the two genes share high structural similarity and are func-tionally interchangeable (Beach et al, 1982). This functional identity of this kinase ap-parently extends across many eukaryotic species and functional complementation of yeast mutants has been a highly successful route to the detection and isolation of the homologous genes in other organisms, hi yeast and muMcelmlar fungi Aspergillus, a single cdc2/CDC28 kinase is required for two critical control points in the cell cycle: Gl/S and G2/M transitions (Nasmyth 1993; Osmani et al., 1994; Piggott et al., 1982). Other members of CDC25-related kinases have been isolated from S. cerevisiae as mentioned earlier. In S. pombe, a cyclin, hsc26, has been discovered which does not form a complex with cdc2 kinase, suggesting that multiple cdc2 members might also be present in this unicellular organism (Orgas et al, 1991). It has become apparent that multi-cellular organisms possess multiple cell cycle regulating kinases highly related to cdc2. CDC2 is mainly needed for entry into mitosis in higher eukaryotes; transitions at early stages of the cell cycle are controlled by other CDC2-related kinases (Meyerson et al, 1992; Solomon, 1993; Van den Heuvel and Harlow, 1993). In human, more than ten cdc2-related kinases, including cdc2 itself, cdk2, cdk3, PSK-J3 (cdk4), PSSALRE (cdk5), PLSTIRE (cdk6), CAK (cdk7), PCTAIRE-1, PCTAIRE-2, PCTATRE-3, KKIALRE and p58-GTA, have been isolated (Meyerson et al, 1992; Tassan et al, 47 1994; Table 1). They share 40% to 83% amino acid sequence identity with human p34cdc2. To initiate analysis of the molecular constituents of the Paramecium cell cycle regulation, I isolated a cdc2 homologous sequence from Paramecium tetraurelia by a combination of polymerase chain reaction (PCR) procedures. The corresponding amino acid sequence exhibited about 50% identity to the cdc2 proteins of other species. The Paramecium a/c2-encoded protein was 11 amino acids longer than S. pombe $34cdc2. There was a single amino acid change in the conserved PSTAIRE region. Southern blot analysis, as well as sequence analysis of another closely related cdc2 cDNA sequence, which was 96% identical to the first complete sequence at the amino acid level over 83 % of the gene, indicates that Paramecium has multiple members of the cyclin-dependent kinase family. 2.2 RESULTS 2.2.1 Isolation of a cdc2-Uke Sequence from Paramecium by PCR Western blot analysis of Paramecium cell extracts with anti-PSTAIRE antibody suggested that the ciliate Paramecium, like all other eukaryotes examined, has cdc2-cdc2 related kinases (Tang et al, 1994). The remarkable conservation of the p34 kinase between highly diverse species allowed me to use the polymerase chain reaction (PCR) to isolate homologous sequences from Paramecium tetraurelia. Degenerate primers from three regions which are highly conserved in other functional CDC2 proteins: AMKKIR, WYRAPE, and GCEFAME were designed for PCR (Fig. 2-3). The most likely codon usage of Paramecium tetraurelia was used to design degenerate oligonu-cleotide sequences to these regions. Genomic DNA was used as template in the PCR reactions. A 450-bp fragment was amplified by the pair of a sense primer from the AMKKIR region and an antisense primer from the WYRAPE region. The PCR reaction with primers from AMKKIR and another antisense primer from the GCLFAEM region 48 yielded a 510-bp product. Both PCR products were purified, subcloned into Bhiescript plasmid DNA and partially sequenced. The derived amino acid sequence of these two PCR fragments were identical in the overlapping region. They exhibited a high similar-ity to the cdc2 proteins of other species, and included the PSTAIRE motif characteris-tic of the cdc2 gene family except for one change out of 16 amino acids, and the kinase domain VI (Hanks et al., 1988) which occupies a long conserved sequence (LHRDLKPQNLL) among different cdc2 proteins (Fig. 2-3). 2.2.2 Isolation and Characterization of Genomic and cDNA cdc2PtA Specific primers were designed from the above PCR fragment for isolation of * 3'- and 5'-end sequences by RACE and RAGE as described in Materials and Methods. Then two primers, one from each end of the gene, were made to isolate the full se-quence from both genomic and cDNA by PCR The resulting PCR products were sub-cloned into plasmid DNA and sequenced. The nucleotide sequence, as well as the pre-dicted sequence of the protein encoded by the gene which is named cdc2PtA, is shown in Fig. 2-1. The ATG initiation codon is likely the real start codon of the Paramecium cdc2PtA sequence isolated. There was a stop codon (TGA) 40 nucleotides upstream of the start codon and there were no other start codons between these two codons. The very A/T rich upstream provides further evidence for this being the start (Fig. 2-1). Fig. 2-1. Nucleotide and deduced amino acid sequences of the Paramecium tetraurelia cdc2 gene, cdc2PtA. The amino acid sequence is shown in one letter code above the genomic sequence. * indicates the stop codon TGA. Q* is glutamine encoded by TAA in Paramecium. The regions upon which the primers were designed for RAGE are un-derlined and that for RACE is double underlined. Two short introns are present in the sequence. Pairs of GT/AG specific to eukaryotic introns are indicated by filled trian-gles. The first stop codon (TGA) before the start codon is indicated by bold italic. The numbers on the right are positions of the last nucleotides and amino acids, respectively. The sequence was submitted to Genbank and given the accession number U1S802. 49 TATGGGTGGTTTGTTTGATTTGAT TGAAAATGGAAAAATAAAAATCGTATATTAATATTTAATATT -66 ATGAAATCTAATAAACTGGAGAAATACGAGAAGAAGGAGAAGCTCGGGGAAGGTACTTACGGAATTGTG 6 9 M K S N K L E K Y E K K E K L G E G T Y G I V 23 TATAAGGCATTGGGTATGCATCCTATTATTATTTGCTATTTAGACCGTAACACTAATGAGTATGTGGCG 13 8 Y K A L A ^ D R N T N E Y V A 36 ATTAAAAAAATCAGACTTGAGTCAGAAGAGGAAGGCATTCCTAGTACTGCCATCAGA GAGATCTCTCTT 20 7 I K K I R L E S E E E G I P S T A I R E I S L 59 CTCAAAGAGTTGAATCATCCAAACATTGTGAAGTAATACTCGTTCTATCTAGAGATTAATGGAGGTTGTG 2 77 L K E L N H P N I V K A ^ L M E V V 75 CATTCAAATAAGAAACTTGTACTGGTCTTTGAGTATTTTGAAATGGATCTTAAGAAATTCCTTGCC CAA 346 H S N K K L V L V F E Y F E M D L K K F L A Q 98 TTCCCCAAAGAGAAAGGC ATGGAACCCGTGATTGTGAAGAGTTTCCTCTATTAACTCCTAAGAGGAATC 415 F P K E K G M E P V I V K S F L Y Q * L L R G I 121 TAAGCATGTCATTAATAAAAAATATTACACCGTGATCTCAAACCTCAAAATCTTTTGGGCTCCAAAGAC 484 Q * A C H Q * Q * K I L H R D L K P Q N L L G S K D 144 GGAATCCTCAAACTTGCTGATTTTGGACTTGCCAGAGCTAGTGGAATCCCTGTTAAAAGTTTCACTCAT 553 G I L K L A D F G L A R A S G I P V K S F T H 167 GAAGTTGTCACACTGTGGTACAGACCACCAGATGTCCTGTTGGGCTCTAAAAACTACAACACCTCAATA 622 E V V T L W Y R P P D V L L G S K N Y N T S I 190 GATATCTGGAGTGTGGGCTGCATCTTTGGCGAAATGTCAAATCTTAAACCTCTTTTTGCTGGAAGCAAT 691 D I W S V G C I F G E M S N L K P L F A G S N 213 GAAACTGATCAATTGAAAAAGATATTCAGAGTCTTGGGAACTCCATCACCTATTGAATATCCTAAATTG 760 E T D Q L K K I F R V L G T P S P I E Y P K L 236 AATGATCTTCCAAGCTGGAAACCTGAAAACTTTGAACAATACCAACCTGACAATCTAGCTAAGTTTTGT 829 N D L P S W K P E N F E Q Y Q P D N L A K F C 259 CCAAGACTTGATCCAGATGGCTTAGACTTATTGGTCAAAATGCTTAAAATTAACCCAGATTAAAGAATT 898 P R L D P D G L D L L V K M L K I N P D Q * R I 282 ACAGCCAAAGCTGCATGTGAACATCCCTTCTTCAAAGAATTACCAGAATAAGTTAAGAAATTATATGTC 9 6 7 T A K A A C E H P F F K E L P E Q * V K K L Y V 305 AACGTCAAGTGAGAATTCATAGATCAAATATATATATATCAAATTTCATAAAACCTTTCA-17 N V K # 1027 308 50 M 1 2 3 4 5 6 0.5 -0.4 -0.34 -0.3 -0.22 _ 0.2 -0.15 _ 0.13 -Fig. 2-2. PCR amplification from the two intron regions. Lanes 1 and 2 corresponding to the PCR products obtained with the first set of primers (PAR-11: 5 '-GCGGATCCATGAAATCTAATAACTGG-3' andPAR-5: 5'-GGTCTAGACTGT ACTAGGAATGCCTTCCT-3') surrounding intron 1; lanes 5 and 6 to those obtained with the second set of primers (PAR-6: 5 '-GGTCTAGAGAGATCTCTCTGCTTAAA GAG-3'; and PAR-12: 5'-GCGAATTCATAGAGGAAACTCTTCAC-3') surrounding the second intron; lanes 3 and 4 to those obtained with set of primers PAR-11 and PAR-12 surrounding both introns. Lanes 1, 3 and 5 are the amplification from genomic DNA; lanes 2, 4 and 6 from cDNA after reverse transcription from total RNA. The minor bands on both lane 4 and lane 6 are from non specific amplification. The size markers (lane M, kilobases, BRL) are indicated on the left of the figure. 51 Comparison between cDNA and genomic DNA sequences shows that Parame-cium cdc2PtA contains two short mteivening sequences, of 30 nucleotides and 22 nu-cleotides, respectively (Fig. 2-1). Both introns contain consensus splicing sites GT/AG, which are specific to eukaryotic introns (Csank et al, 1990); neither contain any in-phase stop codons. Short introns are standard in Paramecium. About 50 introns rang-ing from 20-33 nucleotides have been reported in genomic sequences coding for a va-riety of Paramecium proteins, including phosphatases, kinases, tubulins, and low-molecular weight GTP-binding proteins (Russell et al, 1994; Dupuis, 1992). The sec-ond intron was located at the same place as the second intron of four in S. pombe cdc2 (Hindley and Phear, 1984). PCR amplification using primers encompassing these two intronic sequences from mRNA-derived cDNA and genomic DNA further confirmed that these two short sequences were absent from Paramecium cdc2PtA mRNA (Fig. 2-2). 2.2.3 Similarity of CBC2PTA to Other CDC2-like Proteins A comparison of the derived amino acid sequence with those of the CDC2 pro-teins from other eukaryotes is shown in Fig. 2-3. The predicted CDC2PTA protein contained all 11 domains characteristic of all types of protein kinases (Hanks et al, 1988). The Paramecium ccfc2-encoded protein has 308 amino acids (aa) compared to 288-298 aa from other organisms. The longest known CDC2 protein kinase is the gene cdc2 product encoded by Aspergillus nidulans nimX which is 322 amino acids long with a 15 amino acid insert in the middle region between amino acid 95 and 111 of the gene compared to S. pombe cdc2 (Osmani et al, 1994). Despite the Aspergillus CDC2 hav-ing these additional 15 amino acids, nimXcde2 exhibits over 74 % identity to S. pombe cdc2 sequence at the amino acid level The Paramecium CDC2PTA protein has addi-tional amino acids at both of its N- and C-terminal regions relative to,GDC2 of S. 52 pombe (Hmdley and Phear, 1984). At the amino acid level, the Paramecium cdc2PtA gene is only 41% identical to T. vaginalis (Riley, et al., 1993), 46% to E. histolytica (Lohia and Samuelson, 1993), 60% to P. falciparum (Ross-MacDonald etal., 1994), 56% to CDC28 of S. cerevisiae (Lorincz and Reed, 1984), 53% similar to S. pombe cdc2 (Hindley and Phear, 1984), 59% to that of Z. mays (Colasanti et al, 1991), 55% to CDC2 of D. discoideum (Michaelis and Weeks, 1992) and 54.5% to that of human (Lee and Nurse, 1987). By comparison, the human CDC2 protein is 63% identical to that of S. pombe and 58% identical to that of S. cerevisiae, while the two yeast gene sequence share 62% identity. There is absolute conservation of the ATP-binding site motif (amino acids 16-21, Fig. 2-3), where two important regulatory phosphorylation sites, Thr-14 and Tyr-15 in S. pombe (Thr-19 and Tyr-20 in CDC2PTA), are located. Another regulatory threonine residue (Thr-161/167, Thr-166 in CDC2PTA, Fig. 2-3) was also conserved. The overall similarity between Paramecium CDC2PTA protein and other CDC2 proteins, the presence of all essential catalytic residues, and a compa-rable size to that of all other CDC2 kinases, are sufficient to tentatively designate the isolated Paramecium sequence as cdc2. Fig. 2-3. Ahgnment of the predicted amino acid sequences of CDC2PTA with CDC2 or CDC2-like kinases of various species. The organisms compared are: Paramecium tetraurelia (Pt), Trichomonas vaginalis (Tv), Entamoeba histolytica (Eh), Plasmodium falciparum (Pf), Dictyostelium discoideum (Dd), Saccharomyces cerevisiae (Sc), Schi-zosaccharomycespombe (Sp), Zea mays (Zm) and human (Hs). Dashes indicate iden-tity with Paramecium CDC2PTA sequence, and dots indicate positions of spacers re-quired to rnaxhnally align sequences. Four conserved trytophan residues are indicated by asterisks. Arrows indicate conserved catalytic acidic residues (MacNeil and Nurse, 1993). Filled triangles indicate the residues involved in interaction with other cell cycle components suggested by Norbury and Nurse (1989). The highly conserved domains upon which the degenerate primers were based are underlined. The PSTAIRE region is double underlined. The numbers on the right indicate the position of the last amino acid on each line of different CDC2 protein sequences. 53 Pt Tv Eh Pf Dd Sc § p Zm Hs MKSNK.LEKYEKKEKLGEGTYGIVYKALD - D-SA-H-DM S-FR- . . -QQ V-C--W - TR  VV - - - -HGL-- I V--E-DGG-SR-Q-L K ] -S. . GE-AN-KRL- -V V -E N-Q-V--I V . -E Q - - - V - - I V -E D-T-I - - I V G. .RNTNEYVAIKKIRLESEEEGIP 50 .THIP-DQP-VL-LV-MDL--D 46 .'. .TVC-R L KQ-R-DD 44 ..QN-YG-TF-L K-D 43 .KEKA-GRM--L . .DD-V- 49 LRPGQGQRV- -L D--V- 52 .RHKLSGRI--M D-S--V- 45 . . . K T A - - T I - L Q-D--V- 45 . RHKT-GQV- -M V- 45 Pt Tv Eh Pf Dd Sc £ P Zm Hs STAIREISLLKELNHPNI. PSSV--V-SV--- - T - - -L--VCI--S •-AV-L--K V --I K-S--VP-- -V KDDN-V-DE-NRS M - - G - -R VRLMEWHSNK. KLVLVFEYFEMDLKKFLAQF 99 .LHFRE-ICKDS-IIM-C-FMD N--SKR 94 -E-YDIYLED-.F-Y FCDE--YQ-MSRS 93 -K-YD-I-TK-.R HLDQ L-DVC 92 -S-FD-L-CQN.R-Y LDQ YMDSV 98 YD I DAH--Y FLDL RYMEGI 102 ! LDIL-AES.--Y FLD YMDRI 98 HD E-.RIY LDL MDSC 94 -S-QD-LMQDS.R-Y-I--FLS Y-DSI 94 Pt PK..EKGMEPVIVKSFLYQLLRGIQACHQQKILHRDLKPQNLLGSKDGI.LKLA 150 Tv R -N-DLLR-YAF---C-TYYL-RIG-V---I--E-I-IDRN-L. G 142 Eh S- IPINETR- IV- - I-Q-LAF - -YHQ M I-IN-N-T. I - -G 142 Pf E. . . .G-L-S-TA L N--AY-KDRRV INRE-E.- -I - 142 Ds -A LC-QLI--Y K-LAYS-GHR IDRQ-A. 146 Sc - - . .DQPLGAD K-MM--CK--AY--SHR IN N. G 153 Sp SETGATSLD-RL-QK-T VN-VNF--SRR-I ID-E-N. 151 Zm - E . . . FAKN-TLI--Y 1-H-VAY--SHRV IDRRTNA 145 HS -P. .GQY-DSSL Y I-Q--VF--SRRV IDDK-T.I--- 145 Pt DFnLARASGIPVKSFTHEVVTLWYRPPDVLLGSKNYNTSIDIWSVGCIFGEMSN 204 Tv T-AYCFH-IPYDIE-IK-P--LA-EI-INAPAHG-E I--VIA--AR 196 Eh E LTT-NDRKY-S A-EI ATQ-GGA DAA LI - 196 pf F RKY A M---K-S-T A - - V - 196 D d VS-- -RVY---I A-E S-SVPV-M L- 200 S c F-V-LRAY---I A-E G-Q-S-GV-T--I - - - - A - - C - 207 Sp SF-V-LRNY 1 A-E RH-S-GV A--LR 205 zjh F RT A-EI ARQ-S-PV-V A - - V - 199 H s F---IRVY A-E AR-S-PV I-T--A-LAT 199 f A A A * * * * Pt LKPLFAGSNETDQLKKIFRVLGTPSPIEYPKLNDLPSWKPENFEQYQPDNLAKF 258 T v - N--M-DSQV---1--TE I--EED--DFYKYKINNMPCMKKEK--.FNS- 248 Eh KEE--K-RCKI---F---SQ TEDIWNGVTK--FYLSTFPKWKAK-..LHY 248 pf G T ___p_vS-A---MR---I NSKNW-NVTE--KYD-NFTVYEPLP.WES- 249 D d K S-DC-I--IFR DDSIW-GVTK- -EYVSTFPNWPGQP. YN-1 253 Sc R--I-S-DS-I--IF NEAIW-DIVY--DF--SFPQWRRK- . -SQV 260 S o RS---P-DS-I-EIF---Q NEEVW-GVTL-QDY-STFPRWKRM-.-H-V • 258 Z £ Q P-DS-I-E-F 1 NEQSW-GVSC--DF-TAFPRWQAQ- . - -TV 252 Hs K - - - - H - D S - I - - - F R - - - A NNEVW-EVES-QDY-NTFPKWKPGS.--SH 252 Pt CPRLDPDGLDLLIKMLKINPDQRITAKAACEHPFFKELPEQVKKLYVNVK 3 08 Tv F-GV--ELV--IS QM--EH--N-QS-LN--Y--DVN-NL-HTCME 296 Eh IFHT-ERAV Q--FIYT-EK--S-AD-LK DP-NKPNN 291 Pf LKG--ES-I L LD-N Q-L--AY---NN 288 Dd F--CE-LA IA---QYE-SK--S--E-LL--Y-GD-DTSFF 296 Sc V-S R ID-L-AYD-IN--S-RR-AI--Y-Q-S 298 Sp V-NGEE-AIE-ISA--VYD-AH--S--R-LQQNYLRDFH 297 Zm V-N A S RYE-SK RQ-L--EY--D-EWQ 294 Hs VKN--EN S IYD-AK- -SG-M-LN- -Y-ND-DN-1 - -M 297 54 The major differences between CDC2PTA sequences and other CDC2 se-quences are: one amino acid change (Val to He) within the p34cdc2 hallmark 'PSTATRE' sequence motif which is perfectly conserved among CDC2 kinases of higher eukaryo-tes; only two tryptophan residues out of four were found to be conserved in all other CDC2 proteins; and there were two substitutions in the conserved WYRAPE region (Fig. 2-3). Another region, GDSEID (amino acids 212-217 of S. pombe p34cdc2\ which was essential for G2/M transition (Fleig et al, 1992), is changed to GSNETD in CDC2PTA (amino acids 211-216). This region was not conserved in other lower eu-karyotes such as T. vaginalis, E. histolytica and P. falciparum either (Fig. 2-3). 2.2.4 Paramecium Has Multiple cdc2 Genes Sequence analysis of several cloned RACE-PCR products showed that there were two classes of closely related, but different cDNA clones, that differed particularly downstream of the stop codon. The second one (it is referred to as cdc2PtB) was trun-cated at the 5'-end by about 165 bp as compared to the full sequence of the first gene (cdc2PtA) (Fig. 2-4). It represented 83% of the whole sequence ifcdc2PtB has the same size as cdc2PtA. The sequence identities between these two were 91.5% at the nucleotide level and 97% at the amino acid level. Of 42 altered codons, 36 were silent changes and 6 resulted in amino acid substitutions. In the 3' flanking sequence, 39% of nucleotides differed (Fig. 2-4). Whether these two sequences represented isoforms of the same gene or two different o/c2-related genes is not clear. It is most likely that they are two isoforms of the same gene since Southern blotting showed the same pattern when probed with two antisense oligonucleotides (PAR-14 from cdc2PtA: 5'-GAAAGGTTTTATGAAATTTGATATATA-3'; PAR-15 &omcdc2PtB: 5'-GAGAGAGAAATTTATATGAAATTTAATATT-3') based on the different nucleo-tide sequences located after the stop codon (data not shown). But PCR amplification 55 using a combination of cdc2PtA 5'-end primer and antisense cdc2PtB specific primer (PAR-15) failed, indicating the 5'-end of cdc2PtB is different from that of cdc2PtA. Other slightly different partial cdc2-\ike sequences have also been isolated (Riley, Unfv. Washinston, personal communication; Zhang and Berger, personal communication). Southern blotting analysis showed at least 2 hybridization bands when digested with 7 different restriction enzymes (Fig. 2-5). There was always one band stronger than other bands in any restriction enzyme-digested sample (Fig. 2-5), suggesting that these bands were not just different copies of the same thing, instead, they probably rep-resented different sequences. Other weak bands (3-5) could be seen on extended au-toradiography (4 hr, data not shown). The Southern blotting results further substanti-ates that Paramecium has two or more afc2-related sequence. If this is the case, a fam-ily of o/c2-related protein kinases may be functionally redundant during the Parame-cium cell cycle and it may also explain why a mutation in this class of gene has not been previously identified in Paramecium, although several other cell cycle mutants have been. Fig. 2-4. Comparison of the two different Paramecium cdc2-\Sk.Q sequences (cdc2PtA and cdc2PtB). All sequences are shown in single letter code. The whole nucleotide and amino acid sequences of cdc2PtA are shown in the middle. The nucleotide sequence of cdc2PtB is shown above cdc2PtA nucleotide sequence and identical sequences are indi-cated by dots. The amino acid sequence of cdc2PtB is shown under that of cdc2PtA and dashes indicate identity between these two sequences. * indicates the stop codon. Arrows indicate the beginning of the incomplete cdc2PtB sequence. Q* indicate ghi-tamine encoded by TAA in Paramecium. 56 ATG AAA TCT AAT AAA CIG GAG AAA TAC GAG AAG AAG GAG AAG CIC G3G GAA GGT ACT TAC GGA ATT GIG TAT AAG M K S N K L E K Y E K K E K L G E G T Y G I V Y K OCA TIG GAC CGT AAC ACT AAT GAG TAT GIG GCG ATT AAA AAA ATC AGA CTT GAG TCA GAA GAG GAA G3C ATT OCT A L D R N T N E Y V A I K K I R L E S E E E G I P -> G ..T AGT ACT GCC ATC AGA GAG ATC TCT CTT CIC AAA GAG TIG AAT CAT OCA AAC ATT GIG AAA TTA ATG GAG GIT GIG S T A I R E I S L L K E L N H P N I V K L M E V V G ..C G T.G ..T CAT TCA AAT AAG AAA CTT GTA CIG GIC TTT GAG TAT TIT GAA ATG GAT CTT AAG AAA TTC CTT GCC CAA TIC CCC H S N K K L V L V F E Y F E M D L K K F L A Q F P - - - - - - - - - - - - v - - - - - - - - - - - -.T . .T C . .A . .C . .T T AAA GAG AAA G3C ATG GAA CCC GIG ATT GIG AAG AGT TTC CIC TAT IAA CIC CIA AGA GGA ATC TAA GCA TGT CAT K E K G M E P V I V K S F L Y Q * L L R G I 0 * A C H T A TAA TAA AAA ATA TTA CAC CGT GAT CIC AAA OCT CAA AAT CTT TTG G3C TCC AAA GAC GGA ATC CIC AAA CTT OCT Q * Q * K I L H R D L K P Q N L L G S K D G I L K L A - - - - - - - - - - - - - - V - - - - - - - - -A A GAT TTT GGA CTT GCC AGA OCT AGT GGA ATC CCT GIT AAA AGT TTC ACT CAT GAA GIT GIC AGA CIG TGG TAC AGA D F G L A R A S G I P V K S F T H E V V T L W Y R T T T CT CCA CCA GAT GIC CIG TTG G3C TCT AAA AAC TAC AAC ACC TCA ATA GAT ATC TGG AGT GIG G3C TGC ATC TTT G3C P P D V L L G S K N Y . N T S I D I W S V G C I F G - - - - - - - - - - - - - _ _ _ _ _ _ _ _ _ _ _ A A C.T GAA ATG TCA AAT CTT AAA CCT CTT TTT GCT GGA AGC AAT GAA ACT GAT CAA TTG AAA AAG ATA TTC AGA GIC TTG E M S N L K P L F A G S N E T D Q L K K I F R V L . .T T A.T A C T.. . .T GGA ACT CCA TCA CCT ATT GAA TAT CCT AAA TTG AAT GAT CTT CCA AGC TGG AAA CCT GAA AAC TTT GAA CAA TAC G T P S P I E Y P K L N D L P S W K P E N F E Q * Y - - - T - - - - - - - - - - - - - - - - - - - - -T . . . T.G AC C A CAA OCT GAC AAT CTA OCT AAG TTT TGT OCA AGA CTT GAT CCA GAT G3C TTA GAC TTA TTG GTC AAA ATG CTT AAA Q P D N L A K F C P R L D P D G L D L L V K M L K - - - - - - - - - - - - - - - - - - I - - - -C ..T C ..C ..T ATT AAC CCA GAT TAA AGA ATT ACA GCC AAA OCT GCA TGT GAA CAT CDC TTC TIC AAA GAA TTA OCA GAA TAA GIT I N P D Q * R I T A K A A C E H P F F K E L P E Q * V - - - - - - - - - - - - - D - - - - - - - - - - -G . .A .A TAA.TT.CAT. TITC.CICTCA-19 AAG AAA TTA TAT GTC AAC GTC AAG TGA GAATTCATAQGCAAATATATATATATCAAATTTC K K L Y V N V K * - - - - - - - - * 57 Fig. 2-5. Southern hybridization of Paramecium tetraurelia genomic DNA with cdc2PtA sequence. Twenty fig of genomic DNA were digested with BamHL, EcoRl, HindE, Taq\, Xbal, Xhol, PstI, separated on a 0.7 % agarose geL transferred to Nylon membrane and probed as described in Materials and Methods. The migrations of the molecular size markers (lane M, kilobases (kb), BRL) are indicated on the right of the figure. The film was exposed for 30 min. 58 2.3 DISCUSSION The functional complementation of yeast cdc2ICDC28 mutants has been a highly successful approach for the detection and isolation of the homologous genes in other eukaryotes. However, like most other ciliates, Paramecium cells use only one termination codon. It is impossible to isolate a Paramecium cdc2 homologue by func-tional complementation. With a combination of different PCR approaches, I isolated one full Paramecium cdc2-Vke sequence (cdc2PtA) and 83% of another closely related sequence (cdc2PtB). CDC2PTA was 308 amino acids long. Amino acid sequence identity of Paramecium CDC2PTA to CDC2 kinase of other organisms was around 50%. Southern blotting analysis as well as the isolation of the second incomplete cdc2-like sequence, indicated that Paramecium has multiple CDC2-related members like most other eukaryotes. More than 10 cdc2 related sequences have been cloned from human cells (Meyerson et al., 1992; Tassan et al, 1994). All of them are recognized by anti-PSTAIRE antibody except for KKIALRE and CDK7. Only three (cdkl, cdk2 and cdkS) can complement the ts yeast cdc2/cdc28 mutant at the restrictive temperature. CDC2PTA has much higher similarity to these three CDKs than to other human CDKs. Since human CDK3 does not bind to pl3 , u c ; protein, and a polyclonal antibody raised against cdc2PtA recognized a polypeptide which did not show appreciable pl3 i U C / binding (see Chapter Four), a careful comparison between cdc2PtA and these three CDKs was carried out. However, there was no significant difference among the CDKs, i.e. 54.5% to CDC2, 56% to CDK2 and 54% to CDK3. The structural homology of Paramecium CDC2PTA to CDC2 kinases from other organisms is obvious. Among the five acidic amino acids that are conserved in protein kinases and required for S. pombe cdc2 function (MacNeil and Nurse, 1993), four of them were conserved in Paramecium, and the glutamic acid in domain VIII 59 (Hanks et al, 1988) was substituted by an aspartic acid (Fig. 2-3). This aspartic acid is also present in Plasmodium (Ross-MacDonald et al, 1994). The major regulatory phosphorylation sites (Thr-14, Tyr-15, and Thr-161/167) were all conserved also. An-other phosphorylatable serine reported in vertebrates (Ser-277) was replaced by a threonine (Fig. 2-3). However, this threonine residue is conserved between Plasmo-dium, plant and Paramecium (Ross-Macdonald, 1994; Colasanti et al, 1991; Miao et al, 1993). All these suggest that the isolated CDC2PTA may have the same function and may be regulated like & pombe CDC2 kinase. However, p\3sucl binding sites are not well conserved between Paramecium CDC2PTA, S. pombe CDC2 and human CDC2 (Ducommun etal, 1991a; Marcote et al, 1993). This correlates well with the observation that Paramecium CDC2PTA antibody does not recognize any p I3sucl binding protein (Chapter Four). Nine residues originally thought to be involved in cer-tain CDC2-specific interactions with other cell cycle control elements and conserved in all known functional \>34cdc2 homologues, are absent at the equivalent positions from all other known protein kinases (Hanks et al, 1988, Norbury and Nurse, 1989). Surpris-ingly, only two of the nine were present in Paramecium CDC2PTA sequence (Fig. 2-3). This supports the previous observation that the Paramecium CDC2-like kinases do not need to form a complex with other cell cycle components to become an active his-tone HI kinase (Tang et al, 1994). However, all of the known cyclin-binding sites in S. pombe p34cdc2 and human CDC2 are conserved in CDC2PTA (Ducommun et al, 1991b; Marcote etal, 1993). The EGVPSTAIREISLLKE sequence is the longest stretch of perfect conser-vation of amino acid sequence found across the p34cdc2 functional homologue family in higher eukaryotes (PSTAIRE region, the protein kinase domain HL Hanks etal, 1988; Picard et al, 1990). However, it is not very conserved in lower eukaryotes (Fig. 2-3). In Paramecium CDC2PTA, there is one amino acid change, two changes in Dictyoste-lium CDC2 (Michaelis and Weeks, 1992), four changes in Plasmodium CDC2 (Ross-60 Macdonald, 1994). The PSTATRE region was not at all conserved in Trichomonas or Entamoeba CDC2 (Riley et al., 1993; Lohia and Samuelson, 1993). It has been sug-gested by MacNeil and Nurse (1993) that the precise sequence of the PSTATRE region is not essential for p34"w function, since all mutations in this region with the exception of one (the second glutamic acid) were able to rescue the temperature sensitive cdc2-33 allele. Among those protein kinases with near-perfect PSTATRE sequence is the bud-ding yeast PH085 protein kinase, which has 14 out of 16 matches (Toh-e et al., 1988). One of these two is a valine to threonine change at the same position as CDC2PTA. Until now, cdc2PtA is the only afc2-related sequence which has been isolated from ciliates. It is an essential first step toward molecular analysis of the cell cycle regulation in ciliates. The Paramecium CDC2PTA sequence should also help further define con-served, functionally important regions of the CDC2 protein and its related kinases. 61 CHAPTER THREE GENE EXPRESSION OF CDC2PTA DURING THE C E L L C Y C L E 3.1 INTRODUCTION In S. pombe, cdc2 gene expression does not appear to be regulated in a cell cy-cle stage-specific manner. Cdc2 mRNA levels are constant throughout the vegetative cell cycle and do not decrease as cells enter stationary phase from exponential growth phase (Durkacz et al, 1986). The slime mold Dictyostelium cdc2 mRNA changes not only during the vegetative cell cycle, but also during development. The mRNA level increases just before mitosis. It drops at the beginning of development and increases again before the tipped aggregate stage (Michaelis and Weeks, 1992; Luo and Weeks, personal communication). In muMcellular organisms, cdc2 gene expression drops in terminally differentiated tissues, senescent cells and serum-starved culture cells (Colasanti et al, 1991;Dahon, 1992; Krek and Nigg, 1989; Miao etal, 1993;Richter et al, 1991). In addition, transcription of the human cdc2 gene is cell cycle-regulated and restricted to proliferating cells (Dahon, 1992). The cdc2 transcription is very low before S-phase, increases rapidly during G2 phase and drops again in mitosis. The pro-tein product of the cdc2 gene is held at a constant steady-state level throughout the cell cycle in all organisms examined so far, although it may be depleted in quiescent and differentiated cells, hi mammalian cells, this constant concentration is maintained by a coordinated regulation of protein synthesis and degradation (Welch and Wang, 1992). There have been several previous conflicting reports concerning the cellular lo-calization of CDC2 protein in other organisms. In higher eukaryotes, p34"fc2has been variously reported to be exclusively nuclear, with some specific centrosomal staining at mitosis (Riabowol et al, 1989), or cytoplasmic and strongly perinuclear (Akhurst et al, 1989; Rattner et al, 1990), or both nuclear and cytoplasmic throughout the cell cycle with some specific centrosomal association (Bailley et al, 1989; Rattner et al, 1990). 62 In contrast to these studies in mammalian cells, p34cde2 in S. pombe was reported to be only present as nuclear protein throughout the cell cycle, accumulating in the nucleus in interphase, disappearing during mitosis, and reappearing in anaphase (Alfa et al., 1989; Booher et al., 1989). In addition, the nuclear S. pombe cdc2 fluorescence signal was dependent on the presence of cdcl3 (cyclin B in S. pombe) (Booher et al., 1989). In contrast, the protein product of the Saccharomyces cerevisiae CDC28, is cytoplasmic, possibly associated with some elements of the cytoskeleton (Wittenberg et al., 1987). All these data indicate that subcellular localization of CDC2 protein may vary in differ-ent organisms. It was of interest to determine whether the level of CDC2PTA expression re-flected the replicatfve activity of the Paramecium cell. In this chapter, transcription of cdc2PtA was examined in both exponentially growing and starved cells, as well as at different stages of the vegetative cell cycle. Both elutriation and hand-selection syn-chronized samples were used to examine the cell cycle expression of cdc2PtA. Two polyclonal antibodies, one against the N-terminal GST-CDC2PTA fusion protein and another one against the C-terminal synthetic peptide, were generated. Both cross-reacted with a 36 kDa polypeptide in Paramecium protein extracts on Western blots. These antibodies were used to detennine the protein level in exponentially growing and stationary cells, as well as the steady state protein level during the Paramecium cell cycle. The indirect immunofluorescence technique was used to investigate the subcellu-lar localization of CDC2PTA protein. It was mainly located in the macronucleus in both exponentially growing and stationary cells, but not in micronuclei. 63 3.2 RESULTS 3.2.1 Cdc2PtA Transcripts are Abundant in Actively Dividing Cells Total RNA was isolated from exponentially growing cells and from cells starved for 7 days. In these starved samples, more than 90% of cells were in autogamy (data not shown). Northern blotting analysis showed that a single cdc2PtA mRNA species of approximately 1.3 kb was present in Paramecium cells (Fig. 3-1, B). Unlike its homo-logue in S. pombe, the Paramecium cdc2PtA mRNA was most abundant in actively dividing cells. When the film was scanned using a densitometer, the intensity of the signals from the exponentially growing cells were more than 15 times higher than that of the starved cells (data not shown). The expression in starved cells was almost unde-tectable compared to that of actively dividing cells (Fig. 3-1, B). When the mRNA aga-rose gel was stained with ethidium bromide, there was no difference in rRNA amount, indicating that the same amount of total RNA was used for both samples (Fig. 3-1, A). To determine the relative expression of cdc2PtA transcripts during the vegeta-tive cell cycle, G l cells were selected by centrifugal elutriation, sampled subsequently and assayed at 1 hr intervals. The total RNA was isolated and 10 ug of total RNA were used for Northern blotting analysis (Fig. 3-2, B). Cell synchrony was determined by counting the number of divided cells at one hour intervals. The hand-selected samples from the same culture were compared as a positive control. Nearly 90% of synchro-nized cells divided vvithin a two hour period, from 7.5 to 9.5 hr after elutriation. As it was observed in Chapter One, the synchrony of the elutriated samples was almost the same as hand selected samples (Fig. 3-2, A). Like its homologue in S. pombe, there was no significant difference in the transcription signals between samples and the amount of Paramecium cdc2PtA transcripts remained relatively constant throughout the cell cycle (Fig. 3-2, B, C). 64 Fig. 3-1. Northern blotting analysis of Paramecium cdc2PtA gene expression from starved (right lane) and exponentially growing (left lane) cells. Twenty pg of total R N A were loaded on each lane. A . Ethidium bromide-stained gel of the total R N A before transferring to the membrane. Two major rRNAs (25 S rRNA and 17 S rRNA) are indicated on the left. B. Northern hybridization with cdc2PtA sequence. The above gel was transferred to Nylon membrane, hybridized to Paramecium cdc2PtA D N A probe as described in Materials and Methods. The DIG-11-dUTP-labeled migrations of the size markers (lane M , kb) are indicated to the right. The film was exposed for 4 hr. 65 To further confirm this, an RT-PCR (reverse transcribed PCR) approach was applied to hand-selected samples, since the RT-PCR technique is very sensitive and the synchrony obtained by hand-selection is nearly perfect. Ten samples of 50 dividers were selected under the microscope at one hour intervals, grown at 27°C, and harvested at the same time one hour after the last sample was selected. Total RNA was isolated from each sample, reverse transcribed into cDNA and directly used as templates for PCR The PCR reaction was carried out in the presence of DIG-11-dUTP, so the PCR products can be detected directly by anti-DIG-11-dUTP antibody which is conjugated to alkaline phosphatase. Only 25 cycles were used for each PCR reaction in order to reduce non-specific amplification. To confirm that this procedure can be used for quantitative analysis, different amounts of total RNA were used in the reverse tran-scription reaction and the resulting cDNA was directly amplified by PCR in the pres-ence of DIG-11-dUTP. The amplified products (around 340 bp) were separated on agarose gels, transferred to the membrane and detected by chemiluminescence. As is shown in Fig. 3-3, A and C, the amount of amplified products was generally propor-tional to the total RNA input, ranging from 1 ng to 1 ng used in RT-PCR reaction. Fig. 3-2. Expression of Paramecium cdc2PtA gene during the vegetative cell cycle by Northern blotting. A, Division kinetics. Fifty cells from enriched G l populations were randomly picked right after elutriation, grown at 27°C and the dividers were counted at one hr intervals. Twenty five dividers from the mass culture before elutriation were used as a positive synchrony control Cumulative percentage of cells having reached division is plotted against the time (hr) after elutriation. Open circles, hand-selected control sample; filled triangles, elutriation synchronized sample. B. Northern blotting analysis. The same amount of total RNA (10 ug) was loaded on each lane and probed as described in Materials and Methods. Lanes 1-12 are synchronized samples collected at 1 hr intervals from 0.5-11.5 hrs after elutriation. Cdc2PtA transcript is indicated by an arrow. C. Quantitation of the Northern blot shown in B. 0.5 2.5 4.5 6.5 8.5 10.5 12.5 Time (hr) 2 3 4 5 6 7 8 9 10 11 12 0.5 2.5 4.5 6.5 8.5 10.5 Time After Elutriation (hr) 67 When the same approach was applied to the total RNA samples extracted from hand-selection synchronized samples, the amount of amplified product was not signifi-cantly different from sample to sample during the cell cycle (Fig. 3-3, B, lanes 1-10, C and D). Since the total RNA was prepared from very small samples (only 100 cells), the possibility that PCR products were amplified from contaminated genomic DNA had be eliminated. To do this, 200 ng of genomic DNA were used as templates for a parallel PCR reaction with all other RT-PCR reactions. As it is shown in the last lane of Fig. 3-3, B, the PCR fragments from genomic DNA was larger (about 400 bp) than that amplified from RNA derived cDNA owing to the presence of two short introns in the genomic DNA (Fig. 2-1; Fig. 2-2). This result indicates that RT-PCR products in the synchronized samples were all amplified from Paramecium cdc2PtA mRNA reverse transcribed cDNA and the RT-PCR products should reflect the relative amount of cdc2PtA mRNA in the reaction. This experiment further confirmed that the Parame-cium cdc2PtA transcriptional level does not change during the cell cycle as found for most cdc2 genes in other organisms. Fig. 3-3. Expression of Paramecium cdc2PtA by RT-PCR A. Determination of line-arity between RT-PCR products and RNA input. Total RNA from exponentially grow-ing cells ranging from 1 ng, 10 ng, 50 ng, 100 ng, 200 ng, 500 ng and 1 ug was used in reverse transcription reaction and the whole resulting cDNA pool was used directly for RT-PCR in the presence of DIG-11-dUTP. The PCR products were separated on an agarose geL transferred to Nylon membrane, and detected by anti-DIG alkaline phos-photase conjugate as described in Materials and Methods. The film was exposed for about 30 min. B. Detection of cdc2PtA gene expression during the cell cycle by RT-PCR 50 dividers were hand-selected at one hour intervals, total RNA was extracted and used for RT-PCR as above. Lanes 1-10 are samples from 1 to 10 hr. Lane 11 is the PCR products using 200 ng genomic DNA as templates. The film was exposed for 3 hr. C. Quantitation of the X-ray film shown in A by a densitometer. D. Cell division kinetics and quantitation of the X-ray film shown in B. Twenty-five dividers were se-lected from the same petri dish of culture at the same time as the 10 hr sample, trans-ferred to depression slide, cell numbers were counted at one hour intervals and plotted against time. Arrows indicate the 340 bp fragment amplified from cDNA. 68 C D 69 3.2.2 CDC2PTA Protein is Invariant Throughout the Cell Cycle To investigate Paramecium CDC2PTA expression, two different approaches were used for antibody production as described in Materials and Methods. Both anti-bodies proved to be highly specific at a 1:500 dilution when antiserum was used for Western blotting analysis. They recognized a ~36 kDa polypeptide in Paramecium protein extracts which is the expected size from its encoded amino acid sequence, and no pre-immune sera reacted with this band (Fig. 3-4, A and B; pre-immune sera data are not shown). However, the signal was much stronger when probed with anti GST-CDC2PTA fusion protein serum than anti peptide (CKTLPEQVKKLYVNVK) serum (Fig. 3-4, A and B). In the subsequent experiments, the anti-GST-CDC2PTA fusion protein from the N-terminal of CDC2PTA was used as Paramecium CDC2PTA spe-cific antibody. The specificity of the GST-CDC2PTA fusion protein antiserum was further tested by immunoblotting to GST protein alone and GST-CDC2PTA fusion protein. It only cross-reacted with the GST-CDC2PTA fusion protein and did not react with GST protein (data not shown). When Tetrahymena and S. pombe protein extracts were used, there was no cross-reaction with the equivalent polypeptide (lanes 1 and 3, Fig. 3-4, A and B). The expression of CDC2-related kinases was also tested by im-munoblotting with anti-PSTAJKE antibody. This 16 amino acid PSTATRE sequence is almost perfectly conserved in all known cdc2 genes from higher eukaryotes (Fang and Newport, 1991; Lee and Nurse, 1988) and no exact equivalent sequence has been de-scribed from other protein kinases (Hanks et al, 1988). The specificity of anti-PSTATRE antibody was tested in diverse species. It rec-ognized the S. pombe 34 kDa protein encoded by the cdc2 gene and the equivalent mammalian protein (Fig. 4-1) (Daya-Makin, et al, 1992; Samiei et al, 1992). When anti-PSTATRE antibody was used, signals around 34 kDa were detected from all three organisms (Tang et al, 1994; Fig. 4-1). In Paramecium, the major polypeptide cross-70 GST Fusion Peptide 12 3 M -53.2 i -34.9 11 -28.7 1 2 3 M -53.2 -34.9 -28.7 B Fig. 3-4. Immunodetection by CDC2PTA specific antibodies. Proteins Scorn Parame-cium tetraurelia (lane 2 in A and B), Tetrahymena thermophila (lane 1 in A and B), and Schizosaccharomyces pombe (lane 3 in A and B) were extracted, separated on SDS-PAGE and detected by immunoblotting with Paramecium CDC2PTA antibodies. A. Immunoblotting with antiserum against GST-CDC2PTA fusion protein. B. Im-munoblotting with anti-synthetic peptide (CKELPEQVKKLYVNVK) anti-serum Ar-rows indicate the Paramecium CDC2PTA polypeptide. 71 reacting with anti-PSTAIRE antibody is the polypeptide with the same size as that rec-ognized by both anti GST-CDC2PTA fusion and the synthetic peptide antibodies. The minor one recognized by anti-PSTAIRE antibody, which migrated faster, did not cross-react with specific CDC2PTA antibodies. All these indicate that both antibodies are specific for the Paramecium cddPtA gene. To determine the protein level of Paramecium cdc2PtA during the vegetative cell cycle, Paramecium cells were first synchronized by centrifugal elutriation. The enriched G l subpopulation was sampled at one hr intervals. Cell pellets were directly lysed in 1% SDS containing the proteinase inhibitors described in Materials and Meth-ods and boiled for 3 min. The protein concentration was determined as described in Materials and Methods, SDS-PAGE buffer was then added to IX. Aliquots of each sample containing equal amount of protein extracts were then analyzed by im-munoblotting using affinity purified anti-GST-CDC2PTA fusion protein antibody. Fig. 3-5. The CDC2PTA protein levels during the cell cycle. A. Cell division kinetics. Cumulative percentage of dividers from elutriation synchronized samples (filled trian-gles) and hand-selected positive control samples (open circles), was plotted as a func-tion of cell cycle stage (time after synchronization by elutriation or since the last divi-sion for hand-selection samples). B. Protein levels as a function of cell cycle stages. Elutriation synchronized cells were sampled at one hr intervals from 0.5 to 11.5 hr (lanes 1 to 12), lysed directly by boiling in 1% SDS, then SDS-PAGE sample buffer was added to lx. Equal amounts of total protein from each sample were loaded on the gel and the CDC2PTA protein levels were detected by immunoblotting with affinity purified anti GST-CDC2PTA fusion protein antibody. C. Paramecium proteins from starved (right) and exponentially growing cells (left) were immunoblotted with affinity purified anti GST-CDC2PTA fusion protein antibody. CDC2PTA is indicated with an arrow. The migrations of the prestained molecular markers (kDa, BioRad) are indicated to the right (lane M). 72 A JO 120 1 100 > b 80 4-60 CD c? 40 -»—< | 20 •_ u 4.5 hand-selected • f f 7.5 Time (hr) 10.5 1 2 3 4 5 6 7 8 9 10 1112 M -53.2 -34.9 B 1 2 M -53.2 -34.9 73 Fifty cells from the enriched G l population were also picked up at the beginning of the experiment to determine the cell cycle processing and synchrony, using the hand-selected sample as a positive synchronization control. Again, most of these elutriated cells started division at around 7.5 hr and most of them finished division before 9.5 hr (Fig. 3-5, A). Like the mRNA analysis, immunoblotting showed no stage-specific ex-pression of CDC2PTA and the amount of protein remained approximately constant throughout all stages of the cell cycle (Fig. 3-5, B). This is comparable with CDC2 kinase in general, the activity of which seems to be regulated by post-translational modification and not by their level of transcription or translation. Both exponentially growing and starved cells were also used for immunoblot-ting to determine the protein levels. The same amount of protein extracts were sepa-rated on acrylamide gels, transferred to membrane and immunoblotted with affinity purified anti GST-CDC2PTA fusion protein antibody. As it is shown in Fig. 3-5, C, the 36 kDa signal was stronger in exponentially growing cells than in nutrient starved sam-ples. However, the difference in protein level between actively dividing cells and starved cells was much less than the difference in mRNA levels shown in Fig. 3-1. 3.2.3 CDC2PTA Protein Localizes Mainly in Macronucleus Indirect immunofluorescence was used to examine the subcellular localization of Paramecium CDC2PTA After incubation with anti CDC2PTA antibodies, Parame-cium cells displayed a strong staining of the macronucleus, and no staining of the mi-cronuclei (Fig 3-6, A and B). The occasional staining of the cytoplasm may be just back 74 Fig. 3-6. Immunofluorescent localization of CDC2PTA. Endogenous CDC2PTA was visualized by indirect immunofluoresence microscopy as described in Materials and Methods. B and D, immunostaining with affinity purified anti GST-CDC2PTA fusion protein antibody; A and C, D N A staining with DAPI; A and B, vegetative cell; C and D, cell in autogamy. Arrows indicate the micronuclei. 75 ground, since the signals showed no pattern in the cytoplasm, varied from cell to cell, and some fluorescence was also seen from cells treated with the secondary antibody only (data not shown). Some of the strong fluorescence signals in the cytoplasm are food vacuoles. Uniform results were obtained from all cells in exponentially growing cultures, indicating CDC2PTA may not undergo significant redistribution during the cell cycle. Virtually identical results were obtained using anti-serum or affinity purified antibody. Results were indistinguishable when cells were fixed using 2 % paraformalde-hyde or -20°C methanol (data not shown). When the starved Paramecium cultures were used for immunofluorescence microscopy, the signals were slightly weaker and varied widely between individual cells. In most cells, fluorescence was restricted to macronuclear fragments and the signals were weaker compared to that of exponentially growing cells (Fig. 3-6, C and D). The maconuclear fluorescence signals were not de-tectable when competitive GST-fusion proteins were included in the primary antibody solution (data not shown). I conclude that CDC2PTA is predominantly a macronuclear protein and that there is little if any present in micronuclei 3.3 DISCUSSION In S. pombe, cdc2 mRNA levels do not decrease as cells enter stationary phase from the exponentially growing phase (Durkacze/ al., 1986). However, in multicellu-lar organisms, cdc2 gene expression is very low in differentiated tissues or serum-starved culture cells (Colasanti et al, 1991; Dahon, 1992; Miao et al, 1993; Welch and Wang, 1992). Both mRNA and protein levels of cdc2PtA were low at stationary phase, suggesting that the Paramecium cdc2PtA expression pattern might reflect the status of cell differentiation. The dramatic decrease in mRNA of cdc2PtA as Paramecium cells entered stationary phase was therefore similar to the situation found in the non-dividing 76 cells of adult tissues of multicellular organisms. However, the change in protein level was much less, indicating that CDC2PTA protein may not be degraded immediately when cells proceed to stationary phase, which prevents a dramatic decrease in the level of total CDC2PTA protein. S. pombe cdc2 mRNA levels do not oscillate during the vegetative cell cycle (Durkacz et al, 1986). But in mammalian and unicellular Dictyostelium cells, the cdc2 mRNA level changes while the protein level remains constant throughout the cell cycle (Michaelis and Weeks, 1992; Welch and Wang, 1992). In Paramecium, the level of cdc2PtA mRNA remained relatively constant throughout the vegetative cell cycle, which parallels the expression from the yeast cdc2 gene. The protein levels expressed from cdc2 remain constant during the cell cycle in all eukaryotes examined to date and Paramecium CDC2PTA is no exception. Thus the Paramecium cdc2PtA gene does not appear to be differentially regulated, either at the trancriptional or translational level, during the cell cycle. Two polypeptides cross-reacted with the anti-PSTATRE antibody in Parame-cium protein extracts. The major polypeptide was heavier than S. pombe $34cdc2, roughly 36 kDa (Fig. 4-1; Tang et al, 1994). This polypeptide has the same size as that recognized by cdc2PtA antibody, indicating that the isolated cdc2PtA is the major cdc2-related gene in Paramecium. The minor polypeptide migrated at the same rate as S. pombe p34cdc2 and did not cross-react with CDC2PTA antibody (Fig. 4-1). Paramecium CDC2PTA protein showed up as a doublet in immunoblotting us-ing anti-PSTATRE or CDC2PTA specific antibodies. It is unlikely that this doublet rep-resented two different proteins, since antibodies against the N-tenninal GST-CDC2PTA fusion protein and the C-terminal synthetic peptide, as well as anti-PSTATRE antibody, recognized the same doublet. I assume that this doublet is differ-ent modified forms of CDC2PTA. In other eukaryotes, CDC2 kinase 6an be phosphory-77 lated in a cell cycle-dependent manner at up to four different sites (Gould et al, 1991; Krek and Nigg, 1991). The different phosphorylated forms can be distinguished by their electrophoretic mobility in higher eukaryotes (Choi et al, 1992; Pondaven et al, CDC28 1990). However, band shift of p36 has not been observed in S. cerevisiae, al-though it is regulated partly by phosphorylation (Reed et al, 1988). In Paramecium, I did not observe any obvious band shift throughout the cell cycle, suggesting that, like their homologues in budding yeast, these proteins may not have many phosphorylation sites (Reed et al, 1988). I tested the tyrosine phosphorylation by immunoblotting with 3 different anti-phosphotyrosine antibodies from Upstate Biotechnology Inc.(4G10), Santz-Cruz (PY20) and BRL (GD3CO BRL, Life Technologies, Inc.), but hone of these antibodies cross-reacted with the two Paramecium polypeptides recognized by anti-PSTAIRE antibody or the CDC2PTA specific antibody (data not shown). This suggests that either the Paramecium CDC2 homologues are not regulated by tyrosine phospho-rylation as CDC28 in budding yeast (Amon et al, 1992), or that there was insufficient tyrosine phosphorylation of the CDC2-like proteins for detection by these antibodies, or that the phosphorylation state does not change at all during Paramecium vegetative cell cycle, or that such modifications might have been lost during the protein extract preparation and thus went undetected. Immunofluorescence microscopy of CDC2PTA has provided information con-cerning the cellular distribution of CDC2PTA protein in Paramecium. It was located in the macronucleus, showed no redistribution during the vegetative cell cycle and a slight reduction in signal was observed in starved cells. In S. pombe, v34cdc2 accumulated in the nucleus during interphase, but was undetected at the end of mitosis (Booher et al, 1989). However, the cyclic nature of the CDC2 nuclear fluorescence was not noted in mammalian cells (Riabowol et al, 1989). The detection of p34"fc2 within a variety of domains of the mitotic apparatus in vertebrates including centrosome, kinetochore and mitotic microtubules (Bailley et al, 1989; Rattner et al, 1990; Riabowol etal, 1989), 78 suggests that n34edc2 may play a role in events associated with metaphase and ana-phase. Similarly, cyclin A-CDK2 complexes have been reported to be highly concen-trated at DNA-replication sites as it is required for DNA replication in vertebrates (Girard et al., 1991). The macronuclear distribution of CDC2PTA probably ensures that the CDC2PTA kinase activity will remain near the target site to regulate cell cycle events associated with the macronucleus. The possible association of CDC2PTA with macronuclear DNA synthesis will be discussed in more detail later in the next chapter and in the general discussion. 79 CHAPTER FOUR CHARACTERIZATION OF CDC2PTA KINASE 4.1 INTRODUCTION Under most circumstances, CDC2 or its related kinases are not regulated at the transcriptional or translational level during the cell cycle. Instead, the cell cycle depend-ent oscillation in CDK activity is regulated by complex mechanisms that include inter-action with other regulatory proteins, as well as by phosphorylation/dephosphorylation at both positive and negative regulatory sites. CDKs are typically inactive as monomers, and the initial step in CDK activation is binding to cyclins. The concentration of these regulatory proteins usually oscillate dramatically during the cell cycle and their change is usually correlated with their associated CDK kinase activity. The resulting CDK-cyclin complex has very low kinase activity and is extensively regulated by phosphory-lation. Phosphorylation of S. pombe p34"^ at Thr-167 (Thr-160 in human CDK2 and Thr-161 inhuman CDC2) leads to maximal activation of the CDK-cyclin complex. In some cases, the complex can also be negatively regulated by phosphorylation at Thr-14 and Tyr-15. Removal of phosphate groups from these residues leads to the final activa-tion of the kinase complex. In this chapter, CDC2PTA was further investigated to determine if it acts as a CDK kinase. First, CDC2PTA was tested for binding capacity for S. pombe p I3sucl, which interacts strongly with v34cdc2 both in vivo and in vitro. Unlike the majority of CDC2s found in other eukaryotic groups, CDC2PTA did not show any affinity for the S. pombe p\3sucl protein. Immunoprecipitated CDC2PTA protein accepts histone HI and casein as in vitro substrates. The CDC2PTA-associated histone HI kinase activity oscillated during the cell cycle with maximum activation at the point of initiation of macronuclear DNA synthesis. Both immunoprecipitation of [35S]-methionine labeled Paramecium proteins as well as glycerol density gradient centrifugation, showed that 80 CDC2PTA protein mainly exists as monomers and that the monomers were active with histone HI phosphotransferase activity. 4.2 RESULTS 4.2.1 CDC2PTA Protein Does not Associate with pl3sucl The S. pombe sucl gene was isolated as a high-copy suppressor of cdc2 mu-tants (Hayles et al, 1986). Its gene product, p\3sucl, has been shown in both genetic and biochemical studies to be a negative regulator of mitosis through interaction with cdc2 kinase and complexes containing cdc2 and cyclins (Basi and Draetta, 1995; Bri-zuela et al., 1987; Dunphy and Newport, 1989; Moreno et al, 1989). Purified S. pombe p 13sucI has been used as a high affinity ligand to purify CDC2 kinase from a va-riety of organisms including plants, yeast, frog and HeLa cells (Brizuela et al, 1987; John et al, 1991; Pondaven et al, 1990), owing to its specific and high affinity binding capacity for CDC2 kinase in vitro. To investigate whether CDC2PTA was able to in-teract with p\3sucl, Paramecium protein extracts were incubated with pl3iUc7-coated Sepharose beads, and both pellets and supernatants were immunoblotted with affinity purified antibody against the GST-CDC2PTA fusion protein from the N-terrninal re-gion. As shown in Fig. 4-1, A, there was no difference between the extracts before binding (lane W) and the supernatants after binding (lane S) to p\3sucl beads. The 36 kDa polypeptide (CDC2PTA) stayed in the supernatant and no signal was detected in the pl3*uW pellets (lane P, Fig. 4-1, A). There was a weak band just below the 28.7 kDa marker in the p\3suel pellets, but this band was also present in the whole protein ex-tracts and supernatants, indicating that this is a non specific binding to pl3""7 beads (Fig. 4-1, A). To confirm that pi 3SUC/ beads bind specifically to p34cdc2 kinase, S. pombe protein extracts were used as a positive control. After pl3*uc7 precipitation, the pellets were immunoblotted by affinity-purified anti-PSTAIRE polyclonal antibody. In contrast to the Paramecium extracts, a significant percentage of S. pombe p34cdc2 was pelletable 81 by p 13suc beads. Other PSTAJRE-reactive peptides in both higher and lower molecular weight regions remained in the supernatant. The single band at around 34 kDa which was detected in the pellets is the gene product of S. pombe cdc2 (Fig. 4-1, B). Paramecium and Tetrahymena extracts were also tested with anti-PSTAIRE antibody for p\3sucl binding. As it is shown in Fig. 4-1, C and D, two major polypep-tides from both Paramecium and Tetrahymena cross-reacted with anti-PSTAIRE anti-body. In Parmecium, the major polypeptide migrated at 36 kDa which is the same size as the polypeptide that reacted with anti-CDC2PTA antibody. The minor one migrated at the same rate as S. pombe p34c<fc2. When analysed for pl3sucl binding, only the faster migrating polypeptide was detected in the pellet fraction. Almost all of the 36 kDa polypeptide remained in the supernatants. In Tetrahymena, the major polypeptide migrated faster (36 kDa) and it did not bind to p\3sucl. The minor slower migrating polypeptide (37 kDa) was detected in the pellets (Fig. 4-1, C). The two polypeptides were also detected using a monoclonal anti-PSTAIRE antibody (Fujishima et al., 1992). Roth et al (1991) demonstrated that a 36 kDa CDC2-like kinase was present in isolated Tetrahymena macronuclei using a polyclonal antibody against S. pombe p34cdc2 and this peptide was precipitable with pl3suc' beads. The high degree of specificity of pl3S U ( : / for p 3 4 a ^ has also been illustrated in previous papers (Samiei et al., 1992; Tang et al., 1994). I conclude that the protein encoded by the isolated Paramecium cdc2PtA gene exhibited no affinity for S. pombe pl3 s u c / . Fig. 4-1. P13SUC binding analysis. Protein extracts were incubated with pi3*"' beads. The whole protein extracts (W), supernatants (S) and pellets (P) of the pl3*uc / beads were detected by Western blotting. A. Paramecium tetraurelia protein extracts de-tected by CDC2PTA specific antibody. B. & pombe protein extracts detected by affin-ity purified anti-PSTAJHE antibody. C. Tetrahymena extracts blotted with anti-PSTAIRE antibody; D. Paramecium extracts probed with anti-PSTAIRE antibody. The migrations of the prestained molecular standards (kDa, BioRad) are indicated to the right. 82 83 4.2.2 CDC2PTA Kinase Activity Throughout the Cell Cycle The cdc2PtA sequence contained all of the typical kinase subdomains (Hanks et al., 1988; Fig. 1-3). To assay for kinase activity associated with Paramecium CDC2PTA, protein extracts were immunoprecipitated using either anti-serum or affin-ity purified antibody against the bacterialry expressed GST-CDC2PTA fusion protein. After extensive washing, the immunoprecipitates on protein-A agarose were added to kinase assay reactions containing [y-32P]-ATP, and bovine histone HI, or dephosphory-lated bovine casein, or bacterially expressed GST-Rb protein. The phosphorylated sub-strates were separated by SDS-PAGE, the gel was dried and exposed to X-ray film As shown in Fig. 4-2, casein and histone HI were readily phosphorylated by anti-GST-CDC2PTA fusion protein antibody immunoprecipitated protein (Fig. 4-2, lanes 3, 5, 7, and 9), but not GST-Rb protein (Fig. 4-2, lanes 1-2). When both antibodies were ex-posed to excess GST-CDC2PTA fusion protein from N-terminal of CDC2PTA kinase prior to immunoprecipitation, neither histone HI nor casein phosphorylation was de-tected (Fig. 4-2, lanes 4, 6, 8, 10). The anti-peptide antibody which worked well on immunoblotting did not work in immunoprecipitation (data not shown). Under these assay conditions, there was no auto-phosphorylation associated with Paramecium CDC2PTA protein, since there was no phosphorylated protein detected when the kinase assay was performed without adding exogenous histone HI (lanes 3 & 6, Fig. 4-5). In a separate experiment, Paramecium protein extracts were heated to 70°C for 3 min to inactivate the endogenous kinases and diluted into kinase assay buffer. The CDC2PTA immunoprecipitates were then added, and the mixture was assayed for kinase activity on the endogenous substrates. A 30 kDa protein was phosphorylated by CDC2PTA kinase (Fig. 4-3). At present, it is not clear what is the most efficient sub-strate for CDC2PTA kinase. 84 E tibody E "um+Comp tibody tibody+Comp E um+Comp tibody tibody+Comp c CO (D </) (D c CD c CO CD (/) CD (/) * 1 C CO c CO 2 3 4 5 6 7 8 9 10 Fig. 4-2. Zra v//ro kinase assay. Paramecium protein extracts were immunoprecipitated with anti GST-CDC2PTA fusion serum (lanes 2, 5-6, 9-10) or affinity purified anti GST-fusion antibody (lanes 1, 3-4, 7-8) and assayed for kinase activity. Lanes 1 and 2, with GST-Rb fusion protein as substrate; lanes 3-6, with histone H I as substrate; lanes 7-10, with casein as substrate. In lanes 4, 6, 8, and 10, immunoprecipitation was carried out in the presence of competitive GST-CDC2PTA fusion protein before kinase assay. 85 Fig. 4-3. Endogenous substrate from CDC2PTA. Paramecium proteins were heated to 70°C for 5 min and used as substrates for CDC2PTA kinase which was immunoprecipi-tated from Paramecium extracts. Lane 1, heat-treated proteins incubated with CDC2PTA precipitate; lane 2, immunoprecipitation was performed in the presence of the competitive antigen (purified GST-CDC2PTA fusion protein); lane 3, heat-treated proteins in the kinase assay buffer only. Note a 30 kDa protein was phosphorylated by CDC2PTA kinase in lane 1. If one assumes that the cdc2 homologue plays a role in Paramecium cell cycle regulation, I would expect that the CDC2PTA-associated kinase activity oscillates during the cell cycle, since neither cdc2PtA mRNA nor protein level changes during the cell cycle. Using the above assay conditions, I next examined Paramecium CDC2PTA-associated histone HI kinase activities in synchronous Paramecium cells at various stages of the cell cycle. As described before, synchronized samples were collected by periodically harvesting enriched G l cells obtained by centrifugal elutriation. Fifty cells were selected for division kinetics analysis (Fig. 4-4). All the samples were harvested by brief centrifugation, frozen immediately in a -70°C ethanol bath and stored at -70°C. Proteins were extracted in hypotonic lysis buffer as described in Materials and Meth-86 ods. Equal amounts of protein extracts from each sample were immunoprecipitated with Paramecium CDC2PTA-specific antibody. The antibody-antigen complexes were absorbed onto protein-A agarose and kinase activity assay was performed on protein-A beads using histone HI as a substrate. The incorporation of [32P] into histone HI was determined by scintillation counting. The means of three independent assays were plot-ted against the time after elutriation. The immunoblotting studies in the previous chapter showed that the levels of Paramecium CDC2PTA protein remained approximately constant over the whole course of the cell cycle as observed in other organisms (Fig. 3-5, B). However, the Paramecium CDC2PTA-associated histone HI phosphotransferase activity oscillated during the Paramecium cell cycle. The activity was relatively low in the newly elutri-ated cells, increased immediately at early stages, reached a peak at 2.5 hr, and declined to the lowest level (about 50% of the maximum) from 4.5 to 8.5 hr. After more than 90% of the cells had divided, kinase activity began to increase again and reached a sec-ond peak between 9.5 and 10.5 hrs, and dropped afterwards (Fig. 4-4). These results showed that histone HI kinase activity was cell cycle stage-dependent. In Paramecium, the initiation of macronuclear DNA synthesis (IDS) is one of the earliest cell cycle landmarks and it occurs at 0.25 in the cell cycle. The first kinase activity peak (2.5 hr) coincided with the initiation of macronuclear DNA synthesis dur-ing the Paramecium cell cycle, assuming the cell cycle length is 9.5 hr as is shown in the figure. When the synchronized samples were taken before 2 hr of the cell cycle had elapsed and incubated with [3H]-thymidine labeled bacteria, very few cells incorporated [3H]-thyrrridine. The number of labeled cells increased abruptly at 2.5 hr which corrsponds with the CDC2PTA phosphotransferase activity peak. The second peak was not as high as the first one and it was not sharp (between 9.5 and 10.5 hrs) (Fig. 4-4). This experiment did not have sufficient resolution to de-87 termine precisely which cell cycle stage contributed to this kinase activity increase. Most likely, the kinase activity peak was the result of CDC2PTA activity increase from early stages of the newly divided cells, since most of cells had already divided 1-2 hr before that time. This is also supported by the DNA labeling data which showed a sec-ond peak at around 10 hr. Time (hr) Fig. 4-4. CDC2PTA associated histone HI kinase activity as a function of cell cycle stage (time after synchronization by elutriation). Protein extracts were prepared from elutriation synchronized cells, immunoprecipitated with CDC2PTA antibody (open triangles) or precipitated with vl3sucI (open circles), and assayed for histone HI phos-phorylation. The percentage of the maximum from at least three independent experi-ments was plotted against time. The division kinetics of the elutriated culture as cumu-lative percentage of cells having reached division are also shown (filled triangles) with hand-selected cells as a positive synchrony control (open squares). The percentage of labeled cells by [3H]-mymidine are shown in 5 point asterisks. 88 The p 13JUC bhiding property of the more rapidly migrating peptide, which was recognized by anti-PSTAIRE antibody, allowed its kinase activity to be assayed more precisely. Equal amount of protein extracts from elutriated samples were incubated with pl3sucl beads, then histone HI kinase activity was assayed directly on the pl3sueI beads. In contrast to CDC2PTA activity, the p\3sucI precipitated activity remained at a low level until 6.5 hr, by which time almost none of the cells had divided, increased abruptly after 7.5 hr as cells started dividing, reached its highest level at around 8.5 hr, and dropped slowly afterward to about 55% of its peak level by the end of the experi-ment (Fig. 4-4). The rise in kinase activity appeared 1.5-2 hr earlier than the increase in the number of divided cells. As mentioned before, the physiologically defined point of commitment to division in Paramecium (PCD) occurs about 1.5 hr before fission. The increase in pl3 , u W preciptated kinase activity coincided with the PCD, suggesting that this kinase may be associated with commitment to division in Paramecium. The kinase activity was still above 50% of the peak value after 9.5 hr when more than 90% of the cells had divided. The experiment did not have sufficient resolution to determine pre-cisely when this kinase was inactivated. CDC2PTA phosphotransferase activity was also assayed in exponentially growing and starved cells. Although CDC2PTA protein level and immunofluorescence signals decreased only slightly in the starved cells, the activity was dramatically reduced when the cells entered stationary phase (Fig. 4-5, A). The kinase activity was about 20 times higher in exponentially growing cells than in stationary cells (Fig. 4-5, B). This kinase activity difference is similar to the cdc2PtA transcription (Fig. 3-1), indicating that only the newly synthesized CDC2PTA protein kinase contributes to the CDC2PTA phosphotransferase activity. 89 1 2 3 4 5 6 ^ Histone H1 Fig. 4-5. CDC2PT associated kinase activity in exponentially growing (lanes 1-3 in A; fed in B) and starved (lanes 4-6 in A; starved in B) cells. The same amount of proteins from both samples were immunoprecipitated with anti GST-CDC2PTA antibody in the absence (lanes 1 and 4) or presence (lanes 2 and 5) of GST-CDC2PTA fusion protein, and assayed for histone HI phosphorylation. A Autoradiography of phosphorylated histone HI. Lanes 3 and 6 show the kinase assay without adding exogenous histone HI. B. Scintillation counting. The same amount of protein from each sample was im-munoprecipitated with CDC2PTA antibody and assayed for histone HI phosphoryla-tion as described in Materials and Methods. The mean of three independent assays was plotted in the figure. 90 4.2.3 CDC2PTA May Have Kinase Activity as Monomer In yeasts, plants and animals, p34 ^ occurs both as a monomer and as a com-ponent of a high molecular mass complex, with only the complexed form being active as a kinase. To determine whether Paramecium CDC2PTA was part of a high molecu-lar mass complex, Paramecium cells were first labeled with [35S]-methionine for 2 hr. The protein extracts were immunoprecipitated with crude antiserum against the GST-CDC2PTA fusion protein. The immunoprecipitated protein was detected by autora-diography. As shown in Fig. 4-6, the Paramecium CDC2PTA specific antibody specifi-cally precipitated a 36 kDa polypeptide, which was the same polypeptide identified by immunoblotting. The identity of this 36 kDa protein was further confirmed by im-munoblotting with the Paramecium CDC2PTA antibodies against the synthetic peptide from the C-terminal (data not shown). Other polypeptides (the band below 53.2 kDa marker in Fig. 4-6) co-precipitated with the antibody were not specific, since they were also found in the presence of the competitive GST-CDC2PTA fusion protein (left, Fig. 4-6). Although the autoradiography detection is very sensitive, I found that it was diffi-cult to get a strong radioactive signal without this background by immunoprecipitation. More than 5 times as much the [35S]-labeled protein (-300-500 ug) had to be used to detect this 36 kDa protein by immunoprecipitation than by immunoblotting. This sug-gests that only a very small amount of CDC2PTA was synthesized during a 2 hr label-ing period. The 2 hr labeling time was the most efficient period to incorporate [35S]-methionine. In humans, newly synthesized CDC2 protein is more susceptible to activation by cyclin binding or to activation by phosphorylation or dephosphorylation, and contrib-utes to the MPF (maturation promoting factor) activity (Welch and Wang, 1992). To further search for cellular proteins (especially that are newly synthesized) that might associate with a portion of CDC2PTA protein, extracts of [35S]-labeled cells were frac-tionated by sedimentation through a glycerol gradient and analyzed by immunoprecipi-91 2 & u E Q_ O E O o o O z M -53.2 m -34.9 -28.7 Fig. 4-6. Immunoprecipitation analysis. 35S-labeled Paramecium protein extracts were immunoprecipitated with antiserum against GST-CDC2PTA fusion protein in the pres-ence (left) or absence (right) of the competitive GST-CDC2PTA fusion protein. The immunoprecipitates were separated on SDS-PAGE, dried and exposed to X-ray film for 20 hr. The migrations of the molecular size standards (kDa, BioRad) are indicated to the right. 92 Fraction Number 15 17 19 2123 25 27 29 31 33 M -53.2 t B S A P E A K Fig. 4-7. Sedimentation of [35S]-labeled Paramecium proteins by glycerol density gra-dient centrifugation. The fractions were collected from bottom to top (left to right) of the gradient, immunoprecipitated with anti GST-CDC2PTA fusion serum and detected by autoradiography. Only the odd fractions after fraction 15 are shown here since there are no signals detectable in the earlier fractions. The migrations of the molecular mark-ers (kDa, BioRad) are indicated to the right. The peak of B S A (68 kDa) migrated at fraction 25. 93 tation. Most of the radioactive signals at around the 36 kDa region appeared in the late fractions (fractions 23-29, Fig. 4-7), which were also after the peak of BSA (fraction 25). It is not evident that Paramecium CDC2PTA exists as a complex in the higher molecular range with any other protein components. The band below the 53.2 kDa marker was not present with the 36 kDa protein in the early fractions, confirming again that it was not specifically associated with CDC2PTA. A weak signal between 28.7 kDa and 34.9 kDa markers appeared to be co-present with the 36 kDa protein in frac-tions 23-27. It is not clear whether this protein was specifically precipitated by CDC2PTA antibody. It was not detected when using non-fractionated proteins (Fig. 4-6). The CDC2PTA signal spans a wide range of fractions around the BSA peak (68kDa), suggesting that CDC2PTA exists as both monomers and complexed forms, even though no other proteins were co-precipitated with CDC2PTA when using [35S]-labeled proteins (Fig. 4-6). It is conceivable that proteins associating with CDC2PTA in vivo do so only when activated, and that they are active only at specific stages of the cell cycle, and inactivated rapidly, that their interaction with CDC2PTA could be very hard to detect by immunoprecipitation. Fig. 4-8. Sedimentation by glycerol density gradient centrifugation. The fractions were taken from bottom to top (left to right) of the gradient. The odd numbers were used for kinase assay and even fractions for immunoblotting detection. A. Fractionated Para-mecium proteins were immunoblotted with affinity purified anti GST-CDC2PTA fusion antibody. B. S. pombe fractions were inmunbblotted with anti PSTAIRE antibody. C. Coomassie blue staining of the BSA standard. The peak of BSA, sedimented in parallel, migrated at fraction 24. D. Kinase activity assay. Sedhnented Paramecium proteins were immunoprecipitated with GST-CDC2PTA fusion antibody (filled circles) and S. pombe proteins were precipitated with p 13sucl beads (open triangles), the kinase activity was assayed on protein-A or pl3sucl beads using histone HI as substrate. The mean of three independent assays were plotted against fraction numbers. The percentage of the maximum histone HI phosphorylation was used for comparison since the absolute ac-tivity from Paramecium and yeast was different. 1 94 Fraction Number 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Paramecium I 91 Mb i ' B. S. pombe 120 100 4-g 801 ^ 60 4-Q_ D S. pombe I 1 I 1 I I I 1 I Y 11 15 19 23 27 31 Fraction 95 The above experiments indicated that Paramecium CDC2PTA can be present as both monomers and complexed forms with other low molecular weight proteins. Ge-netic and biochemical studies in S. pombe, as well as biochemical studies in plants and animals, showed that CDC2 kinase must complex with other proteins to form an active kinase (Feiler and Jacobs, 1990; Moreno et al., 1989; Pondaven et al., 1990). To fur-ther nvestigate whether CDC2PTA monomers could function as a kinase, Paramecium protein extracts were fractionated through glycerol density gradient centrifugation. Even fractions were collected for immunoblotting with the Paramecium specific anti-body to assay presence/absence of Paramecium CDC2PTA protein and odd fractions were immunoprecipitated to assay for histone HI phosphotransferase activities. As shown in Fig. 4-8, A, the signals of Paramecium CDC2PTA were apparent in fractions 24-30, which span the range of molecular size after the peak of BSA (fraction 24) which was around 68 kDa (Fig. 4-8, C). This is consistent with the above immunopre-cipitation results when [35S]-labeled proteins were used. Next, fractionated protein extracts were immunoprecipitated with Paramecium CDC2PTA antibody and assayed for ability to phosphorylate histone HI in vitro. As shown in Fig. 4-8, D, the CDC2PTA phosphotransferase activity peak was at approxi-mately fraction 27, which corresponds to the presence of the maximum Paramecium CDC2PTA protein signals when detected by immunoblotting (fraction 28, Fig. 4-8, A). This indicates that Paramecium CDC2PTA kinase may phosphorylate histone HI with-out complexing with other proteins. Since BSA can dimerize under certain conditions and it was distributed from fraction 21 to 27, it is also possible that the CDC2PTA ac-tivity was contributed by complexed forms of CDC2PTA Fig. 4-9. Gel-filtration chromatography of Paramecium proteins. A Immunoblotting with anti-PSTAIRE antibody. B. Histone HI kinase activities of gel-fihered proteins. Filled squares, total activity; open circles, p I3sucl precipitated activity. 96 Fraction Number 44 46 48 50 52 54 56 <36 kDa <34 kDa 17000 - I 15000 -13000 -1 ' > 11000 -< 9000 -CD flio 7000 -5000 -3000 -1000 -3500 29 kDd — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — r 39 41 43 45 47 49 51 53 55 57 Fraction B 97 For comparison, S. pombe extracts were fractionated through a glycerol gradient in parallel with Paramecium proteins, the even numbered fractions were precipitated with p I3sucl beads, boiled in SDS-PAGE buffer and detected for the presence of S. pombe cdc2 kinase using anti-PSTAIRE antibody. The odd numbered fractions were precipitated with pl3*"c7 beads and assayed for histone HI phosphotransferase activity. Unlike Paramecium CDC2PTA signals, S. pombe cdc2 signals were present in a much wider range of fractions from 16 to 30 (Fig. 4-8, B). The majority of S. pombe cdc2 was present in fractions 22-28 which was almost the same range of CDC2PTA The long "tailing" in the higher molecular regions (early fractions) indicates that some of the S. pombe cdc2 protein exists as complex with other proteins. The histone HI kinase assay showed that the maximum S. pombe cdc2 phosphotransferase activity occured in much earlier fractions (higher molecular range). The kinase activity was high from frac-tions 19 to 23, while most of S. pombe cdc2 protein was present from fractions 22-28 (Fig. 4-6, B). This confirms all other reports that S. pombe cdc2 has to form a complex with other proteins to become an active kinase. Gel filtration analysis was also performed at early stages of the project. Crude Paramecium protein extracts were fractionated by Superose 12 gel filtration chroma-tography and 100 pi of each 250 pi fraction was probed with anti-PSTAIRE antibody by Western blotting (Fig. 4-9, A). The signals of the faster migrating polypeptide were apparent in fractions 48-54, which spanned the range of molecular size 29-45 kDa. The signals of the slower migrating peptide were present from fractions 44-54 with the mo-lecular size range of 31-70 kDa, indicating that Paramecium CDC2-like proteins were present predominantly as monomers. To investigate whether the monomers of Para-mecium CDC2-like proteins phosphorylate histone HI, the kinase activity was assayed on 5 pi of each 250 pi of the gel-filtration fractionated proteins. Two kinase activity peaks were observed (Fig. 4-9, B). The highest peak was located between fractions 48-53, correlating with the presence of anti-PSTAIRE cross-reactive material. I can not 98 eliminate the possibility that part of the histone HI phosphotransferase activity may belong to other kinases which happen to have the same molecular weight. The other peak was distributed in fractions 42-44 where very low anti-PSTAIRE immunoreactive signals were detected. It is still possible that the kinase activity was contributed by a very small amount of CDC2PTA that was complexed with other proteins and was not detectable by immunoblotting. The obvious experiment to address this question is to assay for kinase activity using CDC2PTA antibody on the gel-filtration fractionated proteins to see if CDC2PTA signals correspond to the second kinase activity peak. If so, it will indicatethat CDC2PTA can be active as a monomer. Using p I3sucl precipitation, I tested the p 13sucl associated kinase activity in gel-filtered proteins. Most of the activity was confined to fractions 49-54, corresponding with the presence of the lower molecular weight protein detected by anti-PSTAIRE antibody (Fig. 4-9). From this experiment, I conclude that the minor Paramecium CDC2-like kinase was active as histone HI kinase without complexing with other pro- ., teins. There was no kinase activity present in the molecular range higher than 45 kDa after p 13 precipitation (Fig. 4-9). 4.3 DISCUSSION The function of the isolated Paramecium cdc2PtA gene and its mechanism of regulation have been further investigated in this chapter. The S. pombe p \3sucl protein beads have been used for purifying CDC2 kinase from various sources. However, CDC2PTA protein did not show any affinity for S. pombe pl3suclprotein. The two . polypeptides of slightly different electrophoretic mobilities that reacted with anti-PSTAIRE antibody showed different affinity for p 13™w. Only the more rapidly migrat-ing form interacted with S. pombe p I3sucl. There are precedents for this observation. Among the CDC2-like kinases cloned from human cells, five of them were efficiently 99 recognized by anti-PSTAIRE antibody and three (cdc2, cdk2, cdk3) were able to com-plement cdc28 mutants of 5. cerevisiae (Meyerson et al., 1992). Those three also dif-fered in pl3*uc/ binding properties. Only CDC2 could be pelleted completely by p\3sucl beads, CDK3 bound weakly to pl3'u c' beads, and CDK2 was intermediate. However, sequence comparison between cdc2PtA and human cdc2 and cdk3 showed no signifi-cant difference as mentioned in Chapter Two. The second ccfc2-like protein isolated-from Dictyostelium (called crp, ccfc2-related protein) did not bind to pl3suc], even though it has higher similarity to any o/c2-related kinases from human than Dictyoste-lium cdc2 which did bind to pl3sucl (Michaelis and Weeks, 1993; and Weeks, personal communication). The binding property of CDC2PTA to p\3suel is further supported by the amino acid sequence data. None of the six sue! binding clusters were perfectly con-served between cdc2PtA, human cdc2 and 51 pombe cdc2 (Ducommun et al., 1991; Marcote et al., 1993; Tang et al., 1995). Although pl3™ c / interacts strongly with the cdc2 gene product in yeast both in vivo and in vitro, its function is not clear. The inter-action between p34cdc2 and pl3sucl may be involved in the mitotic aspects of p34cdc2 function, since deletion ofsue J leads to a cell cycle arrest with a mixed phenotype and mitotic spindle (Hindley et al., 1987; Booher et al, 1989). Its overexpression can weakly suppress some p34cdc2 mitotic mutants (Dunphy and Newport, 1989; Labid et al, 1995; Moreno et al, 1989). The lack of affinity of CDC2PTA for pl3 i u W may ex-plain why it is not required for the later stage of the cell cycle. The in vitro kinase activity assay of the immunoprecipitated CDC2PTA protein showed that cdc2PtA encoded a functional protein kinase, which accepted both casein and histone HI as substrates. While there are comparatively few proteins that are definitely phosphorylated by CDKs in vivo and in a manner that is relevant to cell cycle progression, there is no shortage of candidate substrates for CDKs in vitro, ranging from cytoskeletal proteins, chromatin associated proteins, cyclins, tumor suppressors, DNA replication factors, and transcription factors (Nigg, 1993; Table 2). Histone HI is 100 thought to be a mitotic substrate and has been a traditional and the most effective in vitro substrate for different CDKs, even for S. cerevisiae CDC28 kinase which appar-ently had maximal kinase activities during G l phase (Wittenberg and Reed, 1988). I used histone HI for all CDC2PTA kinase activity assays. All the kinase activity assays were performed in the absence of cAMP and free calcium to inhibit cAMP-dependent kinase (PKI, a PKA inhibitor peptide was also included) and calchun-dependent kinases, which are two major kinase families that also phosphorylate histone HI in vi-tro. In the previous report (Tang et al., 1994), the histone HI phosphotransferase ac-tivity from whole Paramecium protein extracts showed similar patterns with the second peak occurring earlier. The crude kinase activity was high at early stages after the G l cells were selected by elutriation, reached a peak at around 3 hr, and declined to about 50% of its maximum from 4-7 hr. When dividing cells first appeared, approximately at 7 hr after elutriation, the HI kinase activity had begun to increase again and reached a second peak at 9 hr (82% of maximum level), and then dropped abruptly as the cells completed division (Tang et al., 1994). In addition, hand-selection synchronized sam-ples showed the same pattern of two histone HI kinase activity peaks over the course of the cell cycle as did the elutriation synchronized mass culture (data not shown). Since the kinase activity in crude extracts was at least 10 times higher than CDC2PTA-associated kinase activity when the same amount of protein was used, the two activity peaks from crude extracts could not be attributed to CDC2PTA kinase alone. Further-more, the second CDC2PTA-associated activity peak was about 1-2 hr later than that of crude kinase activity (located at the same point as the pl3sucl precipitable activity), and it did not drop to the lowest level. It appears that the second peak of the kinase activity increase in this study was derived from those cells which had divided and were at the same cell cycle stage as the first peak. Compared to the CDC2-associated kinase activity changes during the cell cycle in yeast and mammalian cells, CDC2PTA kinase activity during the cell cycle was not 101 very obvious. In S. pombe, S. cerevisiae, Xenopus and mouse oocytes, the increase of CDC2 kinase activity is usually as high as 4-5 times before mitosis (or meiotic M phase) (Choi et al., 1991; Creanor and Mitchison, 1994; Fang and Newport, 1991; Moreno et al., 1989; Stueland et al., 1993). While unlike CDC2 kinase activity, which oscillated dramatically during the cell cycle, CDK2 kinase, the most closely related member of CDC2, increased only 2-fold or less in activity as the cell cycle progressed from interphase to mitosis (Fang and Newport, 1991). The kinase activity of another CDK, CDK7 (CAK), did not change at all during the cell cycle, although it regulated vertebrate CDC2 and CDK2 kinase activities which did change during the cell cycle (Tassan et al., 1994). Paramecium CDC2PTA kinase activity increased about 2-fold at 2.0-2.5 hr after cell division, which is roughly the point of macronuclear IDS (which is located at 0.25 of the cell cycle) when the cell cycle length is 9-10 hr. The less dramatic change in kinase activity at this point may be because CDC2PTA kinase uses other substrates in vivo and thus the endogenous kinase activity could not be fully detected by using bovine histone HI as a substrate in vitro. Alternatively, some of its catalytic activity might be lost during immunoprecipitation. Other proteins, which are involved in the formation of initiation complexes at the origin of DNA replication, like SV-40 T antigen and RF-A (replication factor A), have been shown to be phosphorylated by CDC2 kinase (Nigg, 1993). It is not clear how CDC2PTA controls initiation of ma-cronuclear DNA replication by phosphorylating and thereby regulating proteins that are directly or indirectly involved in initiation of Paramecium macronuclear DNA replica-: tion. Without genetic evidence, I cannot conclude that the in vitro kinase activity paral-lels the kinase activity in vivo. In all other organisms investigated to date, maximal histone HI kinase activity of CDK proteins require the formation of a multi-porypeptide complexes. The results in this study suggest this may not be the case in Paramecium. As is shown in Fig. 4-8, CDC2PTA is different from S. pombe cdc2 in terms of complexing with other proteins 102 to become an active kinase. Among the proteins that form complexes with CDK kinases, cyclins have been widely investigated. Various families of cyclins (A to H, and CLNs) have been identified. It has been proposed that distinct cyclins associate with different CDK proteins to form complexes with different substrate specificities that regulate various events within the cell cycle (Pines and Hunter, 1990). Recently, Lees and Harlow (1993) showed that the conserved cyclin box sequence alone was sufficient for binding to and activation of CDC2 kinase. However, no signals were detected in Paramecium extracts by immunoblotting with anti-cyclin box antibody designed from mouse cyclin B (data not shown). It is not known what types of cyclins exist in Para-mecium or other ciliates. In view of conservation of p34crf^-cyclin complexes among fungi, plants and animals, if Paramecium CDC2-like kinases can be active as monomers, it will be very hard to explain. However, microinjection of Tetrahymena (another ciliate) and Para-mecium protein extracts into Bufo and Xenopus oocytes induced maturation of oocytes only in the presence of recipient protein synthesis, and no MPF (maturation promoting factor) activity (MPF induced oocyte maturation does not require protein synthesis) was detectable (Fujishima and Hori, 1989; Fujishima et al, 1992), suggesting that cili-ate CDC2 homologues may not play any important roles, or have different functions in regulation of cell division, or that their enzymatic activity may be controlled through different mechanisms. For example, the closest CDK cousins, MAP (mitogen-activated protein) kinases, which have also been imp heated in the control of cellular events of cell cycle progression and meiosis (Pelech and Sanghera, 1992), do not need to associate with other regulatory proteins to become an active kinase (Shibuya et al., 1992). At least, from Fujishima's observations (1992 and 1993), we can conclude that Parame-cium and Tetrahymena do not have cyclins functionally equivalent to vertebrate B type cyclins, since that the only protein synthesis requirement for the MPF induced oocyte maturation is the cyclin B protein. 103 In other lower eukaryotes, the CDC2 kinase regulatory mechanism is not as "universal" as it is in higher eukaryotes. In S. cerevisiae, dephosphorylation of the key tyrosine (Tyr-19, equivalent to Tyr-15 of CDC2) was not required for CDC28 kinase activation (Amon et al., 1992) and mutation of this tyrosine residue did not affect the dependence of mitosis on DNA synthesis, nor did it abolish G2 arrest induced by DNA damage (Sorger and Murray, 1992; Stueland etal., 1993). In. Aspergillus, entry into mitosis requires the activation of both CDC2 and NTMA kinases, activation of CDC2 kinase alone did not drive cell division (Osmani et al., 1991). Even in human cells, CDC2 showed in vitro casein kinase activity regardless of whether it formed a complex with cyclin or not (Draetta and Beach, 1988). Moreover, the monomeric p34crfc2 of Xenopus was shown to be active as histone HI kinase as long as it remained phosphory-lated on Thr-161 (Lorca et al., 1992). The GST-CDK2 fusion protein has also been shown to readily serve as a substrate for CDK7 (CAK) in the absence of cyclin binding (Poon et al., 1993; Tassan et al., 1994). The human malaria parasite Plasmodium CDC2-like protein kinase has been expressed in an active formin bacteria (Ross-MacDonald et al., 1994), while all other CDKS from higher eukaryotes have to be in-cubated with cellular extracts or cyclin proteins to become an active kinase. Plasmo-dium is also the closest to Paramecium phylogeneticalry. The same observation has also been made for Dictyostelium CDC2-like protein kinase (Michaelis and Weeks, personal communication). While Paramecium cell cycle control resembles other eukaryotes in many aspects, there are also significant differences. In particular, Paramecium has two distinctive nuclei which initiate DNA synthesis at different times and divide by different mechanisms (mitosis vs amitosis). The atypical division by amitosis of the macronucleus could be exceptional for its cell cycle regulation. Even the more typical mitoticalry di-viding micronuclei of Tetrahymena, another ciliate, have linker histone proteins which lack target sites for CDC2 kinase (Sweet and Allis, 1993; Wu et al., 1994), and they 104 are not essential for chromatin condensation (Shen et al, 1995). Thus, ciliates may represent a more primitive or specialized system of the cell cycle regulation. It is possible that a low molecular weight protein may interact with CDC2-like kinases in Paramecium. The 33-kDa CDK5 purified from bovine brain phosphorylates histone HI without association with any known cyclins or pl3™c', but it exhibited high degree of histone HI kinase activity when complexed with a 25 kDa protein which was co-purified with CDK5 (Lew et al., 1992). Cloning and sequencing of the 25 kDa subunit revealed it to be a proteolytic derivative of a novel 35 kDa protein, p35. The bacteriahy synthesized CDK5 can be fully activated by bacteriahy expressed partial or full length p35 (Lew et al, 1994; Tsai et al, 1994). The D-type cyclins also have low molecular weight (33-35 kDa) and have been shown to associate with and activate CDK2, CDK4, CDK5 and CDK6 (reviewed by Hunter and Pines, 1994). Several low molecular mass proteins at around 20 kDa have been found to associate with CDKs and inhibit their phosphotransferase activity (reviewed by Elledge and Harper, 1994). The apparent molecular mass of Paramecium CDC2PTA by glycerol density gradient analysis at around 70 kDa makes it likely that it may associate with a low molecular weight protein (Figures 4-7, 8, & 9). Whether CDC2PTA has to form a complex with other low molecular weight proteins to become an active kinase is not clear. At least I can conclude that CDC2PTA kinase behaves differently from S. pombe cdc2 kinase in terms of forming an active kinase complex (molecular weight of the complex). If there is a kinase complex formed between CDC2PTA and other regulatory components, the associated partner should have lower molecular weight than that of S. pombe cdcl3. It is also possible that the weak interaction between CDC2PTA and other proteins might be lost during immunoprecipitation or glycerol density centrifugation or gel filtration. 105 GENERAL DISCUSSION AND PERSPECTIVES Among the cell cycle control genes identified in yeasts, cdc2ICDC28 is the only regulatory factor that is required at two critical control points in the cell cycle: first in late G l when cells commit to DNA replication and later again in G2 before mitosis where cells become committed to division. The conservation of this central control system by this kinase and its related members of the CDK family in eukaryotes occurs from fungi to green algae, plants and animals including humans. However, little is known about ciliate systems. Compared to other eukaryotes, the ciliate cell cycle is unique, especially that of the macronucleus. For all other eukaryotes, one of the most important events in the cell cycle is the DNA replication. Careful controls ensure that replication is confined to a specific phase of the cell cycle (S phase). This control is properly coordinated with other cell cycle events in order for replication to occur only once per cell cycle. However, in Paramecium, precise doubling of macronuclear DNA content does not occur in most cells. Instead, all cells replicate some macronuclear DNA, but some cells synthesize litle DNA while others synthesize lots of DNA For example, macronuclear DNA content does not double in cells with a larger amount of macronuclear DNA than average macronuclei, while cells with less than the average DNA content increase their DNA content greater than two-fold in a single cell cycle. The macronuclear DNA synthesis period occupies most of the cell cycle. The macro-nuclear division is relatively imprecise and two daughter cells typically receive 5-10 % more or less macronuclear DNA from the mother cell. Furthermore, Tetrahymena cells with sufficiently high initial macronuclear DNA content enter cell division regularly without replication of macronuclear DNA. In contrast, micronuclear DNA replication and division are more typical of other eukaryotic cells. Study of the cell cycle in ciliates and other lower eukaryotes at the molecular level will provide critical tests of the pro-106 posed ubiquity of common mechanisms as well as revealing some novel and interesting ways of achieving common biological ends. The identification of cdc2ICDC28 homologues in Paramecium is the first step toward the understanding of the molecular basis of cell cycle control in ciliates. So far, this is the only cdc2 related gene that has been isolated from ciliates. The striking overall similarity of the Paramecium cdc2PtA protein with members of CDK ser-ine/threonine kinases, and, in particular, the presence of the EGIPSTAIREISLLKE motif (in Paramecium, the first isoleucine is changed to valine), which is typical of the CDK kinases, leaves no doubt that the isolated Paramecium sequence belongs to this family. However, it is very hard to achieve the expression of Paramecium genes in yeast, and the isolated Paramecium cdc2PtA gene could not be easily tested by func-tionally complementing yeast cdc2lcdc28 mutants. Therefore, I was not able to estab-lish whether or not Paramecium cdc2PtA functions in vivo as a true cdc2 homologue, or if it should be regarded as yet another cdc2 look-a-like of unknown function. Im-munoblotting results with anti-PSTAIRE antibody and Southern analysis, as well as the isolation of the second incomplete cdc2-h\e sequence, suggest that multiple members of the a/c2-related gene family are present in Paramecium cells. The minor one which cross-reacted with anti-PSTAIRE antibody (which has affinity for p I3sucl and has the highest activity at the commitment to division) is probably the cognate of Paramecium CDC2. Although the amount of both mRNA and protein remained relatively constant over the course of the cell cycle, CDC2PTA-associated histone HI phosphotransferase activity oscillated. The two histone HI kinase peaks of the crude protein extracts pre-pared from both hand-selected and elutriated samples were located at the same posi-tions as the two critical control points (IDS, initiation of DNA synthesis, and PCD, point of commitment to division) in the cell cycle (Tang er al., 1994). We cannot con-clude that the histone HI kinase activity peaks were solely the result of CDC2-related 107 kinases. But, in other eukaryotes, the same two transition points are controlled by CDC2 or related kinases. I suggest that the two histone HI kinase activity peaks were contributed partly by CDC2-like protein kinases. Paramecium may have different CDC2-related kinases activated at different points during the cell cycle. If so, the iso-lated cdc2PtA is most likely required for the early stages as its associated kinase activity increased at IDS of the cell cycle and it did not bind to pl3 J u c / whose action is thought to occur in the later stages of the cell cycle. I cannot eliminate the possibility that it may also be needed at other points in the cell cycle, because S. pombe cdc2 kinase activity was very low at early stages of the cell cycle and S. cerevisiae CDC28 kinase activity was mainly detected in G l and S phases, although cdc2ICDC28 is required for both Gl/S and G2/M transitions (Weittenberg and Reed, 1988; Moreno et al., 1989). In human, CDK1, CDK2, and probably CDK3, all appear to participate in cell cycle regu-lation. The identification of the equivalent members have also been described from other species. The presence of the multi-members of the CDK gene family suggest that they may have separate and distinct function in the control of cell growth, division, dif-ferentiation and/or development. So far, another CDC2-like kinase which is detected by anti-PSTAIRE antibody and is precipitable by p 13""' beads, has been shown to be associated with the commit-ment to division in the Paramecium cell cycle (Fig. 4-1, C; Fig. 4-4). The rise in histone HI phosphotransferase activity associated with p\3'ucl beads appeared 1.5 hr earlier than the increase in number of divided cells. As mentioned in the General Introduction, the physiologically defined point of commitment to division (PCD) occurs about 90 min before fission. The pi3 s u e l precipitated histone HI kinase activity increase occurred at the same point as PCD, suggesting that the histone HI kinase activity of the other faster migrating CDC2-like protein recognized by anti-PSTATRE antibody was associ-ated with commitment to division in Paramecium. Therefore, there are at least two CDC2-like proteins in Paramecium, the major one which has been isolated in this study 108 may be involved in macronuclear DNA synthesis initiation and the other one in cell di-vision. Isolation of other cdc2-h\Q genes from Paramecium will help to demonstrate whether the same cdc2 molecule is involved in regulation of initiation of DNA replica-tion and nuclear division of the two functionally and structurally distinctive nuclei pres-ent in the same cytoplasm Perhaps the most unexpected result in this study is that monbmeric cdc2PtA may be active as a kinase. The S. pombe cdc2 gene is not only required for the onset of S phase and mitosis, but is also necessary for the temporal order of the two events, since specific mutations of the cdc2 gene result in DNA replication occurring without intervening mitosis and in entry into mitosis when DNA replication is blocked (Broeck et al, 1991; Enoch and Nurse, 1991; Hayles etal, 1994). In particular, S. pombe cells deleted for cdcl3 (cyclin B in fission yeast), which have no p3Acdc2-p56cdc13 complex, undergo repeated rounds of DNA synthesis and become highly polyploid, with DNA contents of 32 C (copy numer of genomic DNA, e.g, a diploid cell has 2 C DNA con-tent) or above (Hayles etal, 1994), suggesting that the presence of this complex de-fines a cell as in G2 and that its absence defines in Gl . It also was reported that no cy-clin B was detected during Drosophila embryogenesis, where repeated rounds of DNA replication occur (Lehner and O'FarreL 1990). The polyploid macronucleus of Para-mecium may not need a precise control of nuclear division, it may only need to control initiation of DNA replication and the CDC2-]ike kinase in Paramecium may not need a cyclin-like protein partner to activate it. CDC2PTA is located in the macronucleus, exhibits high kinase activity at initiation of macronuclear DNA synthesis, does not bind to \>\3sucl, and may have kinase activity as a monomer, which suggests that cdc2PtA is just such a macronuclear DNA synthesis regulator and may not need a cyclin partner. There is evidence in ciliates that phosphorylation target sites of different histone proteins for different kinases are found in two functionally distinct nuclei (micronucleus divides mitotically, while macronucleus divides amitotically without chromosome con-109 densation). A cdc2-hke kinase of 36 kDa in another oligohymenophoran ciliate, Tetra-hymena thermophila, has been demonstrated in isolated macronuclei by immunoblot-ting with an anti-yeast p34"fc2 polyclonal antibody and possessed histone HI kinase activity (Roth et al., 1991). The Tetrahymena 36 kDa protein also binds to pl3™ c /. Using a monoclonal anti-PSTAIRE antibody, two major cdc2 homologues of 35 kDa (the same one reported by Roth et al, (1991) and 37 kDa, were identified from Tetra-hymena (Fujishima et al, 1992 & 1993). Whether these ccfc2-like proteins in Tetrahy-mena play any role in cell cycle regulation is not known. In ciliates, macronuclei contain a Hl-like linker histone with growth/division-associated phosphorylation characteristics typical of that histone class (Allis and Gorovsky, 1981). It has been shown that Tetra-hymena macronuclear HI is phosphorylated by a p34c</c2-like kinase that is immu-nologically and enzymatically related to mammalian p34"fc2 kinase (Roth et al, 1991). The function of histone HI phosphorylation by p34cdc2 kinase in Tetrahymena thermo-phda and perhaps in all other ciliates must be specific to amitotic macronuclei. In contrast to macronuclei, micronuclei do not contain typical HI linker his-tones. Instead, it has four distinct linker histone polypeptides, refereed to as a, (3, y, and 5 (Allis et al, 1984; Wu et al, 1994). Phosphorylation of these linker histones are thought to be involved in micronuclear chromosome condensation during mitosis which does not occur in the macronucleus. However, none of them contains a recognition site for p34edc2 kinase. Surprisingly, they contain at least one phosphorylation site for cAMP-dependent kinase (PKA) and all of them are phosphorylated by PKA but not by CDC2 kinase (Sweet and Allis, 1993; Wu et al, 1994). It has been suggested that his-tone phosphorylation by either or both kinases acts to promote transient chromatin de-condensation, allowing access of factors necessary to program the genome for cell cy-cle regulated activities such as transcription, DNA replication, or chromosome conden-sation, and that, in the absence of those factors, linker histone dephosphorylation ac-tually facilitates and or stabilize chromosome condensation (Roth and Allis, 1992). Di-110 rect examination on phosphorylation of the purified Tetrahymena linker histones by the isolated Paramecium CDC2PTA kinase may provide more direct evidence for the above hypothesis. Great progress has been made on cell cycle regulation by CDC2 and its related kinases in yeast and mammalian system However, Paramecium and other ciliates rep-resent a very special eukaryotic group. In particular, the differential control of DNA synthesis among different nuclei would be of great interest for cell cycle study. During the sexual stage of ciliates, micronucleus DNA synthesis is controlled to keep the 2C-4C cycle of DNA amount coupled with the cell division. In the newly developed ma-cronuclear primodia (anlagen), DNA synthesis continues until the completion of the highly polygenomic mature macronucleus. In the old macronuclear fragments, on the other hand, DNA synthesis is suppressed and the DNA is slowly autorysed. How these different processes are regulated without confusion within the same cell is of great in-terest. It has been shown in Paramecium that at the restrictive temperature the ccl mutation blocks DNA synthesis in the macronucleus, while in the micronuclei DNA replication proceeds normally. In addition, the ccl mutation has no effect on the micro-nuclei during the sexual pathway, suggesting a difference in the regulation of DNA synthesis in the two types of nuclei (Adl and Berger, 1994). Gene disruption by ho-mologous recombination and antisense has been widely applied to elucidate the gene function in other systems. Vectors with various promoters and selection markers have been developed to transform Tetrahymena cells and have been routinely used for gene b'-J knockout in Tetrahymena (Gaertig et al., 1994; Kahn et al., 1993). A similar gene dis-ruption system in Paramecium is under development (Meyer et al., unpublished data). It should be possible to apply these expression vectors (or their modified forms) in Paramecium to further elucidate the gene function of cdc2PtA during Paramecium cell cycle control I l l SUMMARY Paramecium is a useful eukaryotic model to study such diverse cellular activi-ties as gene amplification, genome rearrangement, cell motility, surface antigen switch-ing, and nuclear differentiation. Its cell cycle is unique when compared to other types of eukaryotes owing to the presence of two distinct nuclei in the same cytoplasm In par-ticular, the amitotic mode of macronuclear division which produces non-identical prog-eny is atypical and this could reflect unsual cell cycle control progress. This work was initiated in order to further understand how the Paramecium cell cycle is regulated at the molecular level. It describes the first isolation of Paramecium tetraurelia genes encoding protein homologous to the cell cycle regulating components of the other or-ganisms. Owing to the absence of a means of obtaining synchrony in mass cultures for biochemical procedures, little is known about the molecular basis of Paramecium cell cycle regulation. In this thesis, I developed an elutriation selection method that pro-duced a relatively pure population of early G l cells. Using this method, the first 10-12 % of elutriated cells were well synchronized and proceeded into the cell cycle as newly divided G l cells. The fact that Paramecium cannot be grown to high density, that it has two distinctive nuclei which replicate their DNA and divide at different times during the cell cycle, and that two nuclei divide by different mechanisms (mitosis vs amitosis), make it impossible to synchronize Paramecium cells by blocking cells at S- or M-phase with DNA synthesis inhibitors or anti-mitotic reagents. The synchronization by cen-trifugal elutriation developed in this thesis makes the molecular and biochemical analy-sis of cell cycle regulation in Paramecium possible and it is the only efficient way to synchronize mass Paramecium cultures. The synchrony obtained by this method is as good as the frequently used hand-selection technique. Throughout this'research project, 112 the elutriation synchronization had been used to collect cell cycle staged samples for molecular and biochemical analysis. A cdc2 homologous sequence (cdc2PtA) from Paramecium tetraurelia was isolated by PCR procedures. It encodes a protein of 308 amino acids. It shares 53%-60% amino acid sequences with CDC2 from other higher eukaryotes. All of the phos-phorylation sites required for the catalytic kinase activity of CDC2 in other organisms are present in CDC2PTA. There is one amino acid change in the conserved PSTATRE region. Southern blotting with 7 restriction enzymes provided evidence that Parame-cium has multiple cdc2-\jke genes. This is further supported by the isolation of a second closely-related partial cdc2-hks sequence and the presence of a second PSTATRE reac-tive polypeptide. Northern blotting analysis indicated that cdc2PtA mRNA decreased dramatically when cells enter into stationary phase from exponentially growth phase. Both Northern blotting analysis using elutriation synchronized samples and the sensitive RT-PCR de-tection of hand-selected samples show that the cdc2PtA gene expression was constant throughout the vegetative cell cycle. The first 1/3 o£cdc2PtA was expressed in E. coli as a GST fusion protein and purified. The recombinant protein and a 15 amino acid peptide from the C-terminus of cdc2PtA were used to raise antibody in rabbits. Both anti-serum cross-reacted with a 36 kDa protein from Paramecium extracts. CDC2PTA protein level only decreased slightly in stationary phase cells and there was no change between different time point samples of the synchronized cells. Using indirect immunofluorescence, Paramecium cells were stained with affinity purified anti GST-CDC2PTA antibody. The fluorescence signals were present in the intact macronucleus of the exponentially growing cells and the macronuclear fragments of starved cells which are in autogamy. Little or no signals were detected in micronu-113 clei. Like its protein level detected by Western blotting, the CDC2PTA fluorescence signal drops slightly in stationary phase cells. CDC2PTA was tested for its ability to bind to & pombe y\3sucl. Unlike most CDC2 kinases from other organisms, CDC2PTA did not show any affinity for p Usucl which binds tightly with \>34cdc2 from various eukaryotes. When anti-PSTAIRE anti-body was used, a second faster migrating polypeptide was detected at around 34 kDa in Paramecium protein extracts. This minor CDC2-like protein showed affinity for Vl3sucl. Using the specific CDC2PTA antibody, Paramecium proteins were immuno-precipitated and assayed for kinase activity in vitro. The CDC2PTA precipitated pro-tein phosphorylated histone HI and casein which inidcated that the isolated cdc2PtA gene encoded a functional protein kinase. When histone HI kinase activity was assayed on the elutriation synchronized samples, it oscillated during the vegetative cell cycle. The kinase activity was low for the newly elutriated cells and increased as cells pro-gressed into S phase. The kinase peak was at 2.5 hr after the elutriation. This CDC2PTA-associated kinase activity increase reasonably corresponded to the macro-nuclear IDS of Paramecium, and implied an association between CDC2PTA kinase and events of macronuclear DNA synthesis. The pl3*uc/ precipitated activity was low at early stages of the cell cycle and increased at the same time as the point of commitment to division which was 1.5 hr before cell division. Immunoprecipitation of [35S]-labeled proteins showed that CDC2PTA was pre-sent principally as a monomer. This was further confirmed by glycerol density gradient centrifugation and gel filtration. 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Cell 75: 155-167. 127 Appendix I: Single and 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 Ghi Glutamine Q Gin Glycine G Gly Ffistidine H His Isoleucine I Re Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Tip Tyrosine Y Tyr Valine V Val -128 Appedix JJ: Nucleotides Single letter Code Abbreviation Nucleotide A dATP Deoxyadenosine 5'-triphosphate C dCTP Deoxycytidine 5'-triphosphate G dGTP Deoxyguanosine 5'-triphosphate T dTTP Deoxythyrnidine 5'-triphosphate 

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