PLACE IN RETURN BOX to roman this checkout from your record. To AVOID FINES Mum on or baton duo duo. DATE DUE DATE DUE DATE DUE U" k' i 'l "'1 WLWWI |L___J| gLf MSU I: An Afflmdivo AdioNEqual Opponunlty Immion NUTRITIONAL AND CEENOTACTIC SIGNALS INACTIVRTE THE EXPRESSION OP A GROWTH-SPECIFIC GENE EARLY IN DEVELOPMENT IN 2193125221119 we“: as by Randy nasean u. Kassanain' A DISSERTATION Submitted to niohigan state University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1990 NUTRIEIOIIL‘IID OIEIOIICTIC SIG-LLB IIICIIVISI III EXPRESSION OP.I GROIIE-SPECIPIC Eflflfl ELIE! IN'DBVELOPIEI! rummaging b! Handy nassan I. Kassanain The cellular 811-0 mold. W W. is a useful model for studying the regulation of growth-specific gene expression because cells cease division while they differentiate on an agar surface. A com library made to vegetative cell transcripts was used to identify transcripts preferentially expressed in vegetative cells. We are studying the mechanism whereby the mRNA for one of these (D2) falls during aggregation when starvation, aggregation specific cell contacts and diffusible factors like cans are present. :n gitrg transcription reactions performed with.nuclei isolated from vegetative amoebae and from cells developing in suspension indicates that starvation, the trigger for the developmental program, inactivates the b2 gene transcription early in development. However, the D2 transcript levels stay high during suspension development and do not fall as a result of starvation or aggregation-specific cell contacts. Rather cells must be deprived of amino acids and cAMP administered to SOnM periodically in pulses, to mimic cAnP signal-relay in aggregation. This effect can be blocked.with reagents that prevent can? from binding to the cell surface receptor in aggregateless mutants known to be defective in a Gcz protein. Experiments with membrane-permeable chap analogues suggest that the loss of D2 transcript levels does not require a rise in the intracellular can? levels. In addition proper regulation of the D2 gene depends on cell proliferation, because blockage of the cell cycle with caffeine or incubation of temperature sensitive growth mutants at a restrictive temperature caused an inappropriate reduction in D2 mRNA levels. I found evidence for both non-specific and specific degradation of D2 mRNA by use of inhibitors: non-specific degradation does not require protein synthesis but specific degradation requires new RNA synthesis. Overall these results indicate that the D2 gene is regulated at the transcriptional and possibly the translational level. Although some of the extracellular signals have been found, the intracellular signal and.mechanisms that trigger a specific degradation of 02 transcripts remain to be determined. DEDICATION Il'his Dissertation is dedicated to my wife, Patma, and my mother, Amena, for their support, sacrifices, patience and understanding throughout my doctorate degree work. iv ACKNOHLEDGHENTS I would like to thank my major advisor, Dr. Hill .1. Kopachik, for his support, suggestions and cooperation which were necessary for the successful completion of this research. Also, I would like to extend my special appreciation for my Doctoral committee consisting of Dr. Neal Band, Dr. James 'rrosko and Dr. James Atkinson for their guidance and assistance during my research. TABLE OF CONTENTS Page LIST or Precurs......................................... ix INTRODUCTION............................................ 1 The life cycle...................................... 3 The organisation of the genome...................... 4 Gene expression during growth and development....... 5 Physiological signals regulate gene expression...... 6 starvation.......................................... 7 Cell contacts....................................... 9 Cyclic AKP.......................................... 11 Control of gene expression.......................... 17 Objectives and organisation of the thesis........... 21 MATERIALS AND NETHODS................................... 24 nictyostelium 8trains..................... ..... ..... 24 Growth conditions................................... 25 Developmental conditions............................ 26 Pulsing with chP................................... 27 Plasmid preparation................................. 27 Large scale plasmid preparation..................... 27 Amplification of the plasmid........................ 27 The cleared lysis................................... 27 Isolation of plasmid in CsCl gradient............... 28 vi Page Removal of CsCl and precipitation 0‘ th. pl‘Md m.......................O..00....C. 2’ Small scale plasmid preparation..................... 30 Vertical rotor preparation of plasmid............... 3o Isolation of inserts from an agarose gel............. 31 misolation........................................ 35 The guanidinium thiocyanate method................... 35 The phenol/chloroformmethod......................... 37 Northernblot analysis............................... 37 Oligolabeling........................................38 Nuclear transcription assays......................... 40 Nuclei isolation..................................... 4o InVitro transcription............................... 41 ehmcalsOOOOOCO0.000.000...0.0.0.0.000...00.0.0.0...‘2 RESULTBOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO‘S 2. The expression of the D2 and D29 genes............... 43 The physiological signals(s) that regulates D2 gene expression......................... 61 The effect of suspension development and cell-cell contacts............................... 62 The effect of elevating the extracellular or the intracellular cAnP levels.................... 65 3. Oscillation of extracellular chP levels is required for D2 gene expression.................. 72 III. Cyclic AMP signals that inactivate D2 gene expression are transduced through cell-cell surface receptors......................... 82 vii Page 1. Activation and deactivation of the cell surface receptor is necessary for D2 gene inactivation........................... 82 2. signal transduction mutants show defective responses................................ 85 IV. Levels of D2 gene regulation....................... 89 l. Transcriptional and post-transcriptional regulation of the D2 gene.......................... 89 DISCUSSIO’OOOOOOOOOOOOOOOOOOOOOOOO...0.00....OOOOOOOOOOO 93 LIST OF REFERENCBBOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO00...... 125 APPENDIx...0.0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO00.0.0142 viii FIGURE 1. 3A. 33. 4A. 43. CC. 4D. 4E. 4F. JG. 5. 6E. 7A. LIST OF FIGURES Page Dovelopnontal 63010 of Win mm. - 3 The pcD-x plasmid used to construct th‘ an“ library.OOOOOOOOOOOOOOOOOOOOOOO0000...... 33 The expression pattern of the growth gene (D2) and the early developmental gene (D29) in XPss strain.during normal development on.agar.......... 44 The ethidium.bromide stained blot of 3A to show equal amounts of RNAuwere loaded.................. 44 The expression pattern in vegetative amoebae of two tsg mutants (XP95 and n32?) incubated at 219C and after shifting an identical culture to 27°C for18hours......OOOOOOOOOOOOOOOOOOOOOO.COO...COO ‘9 The effect of caffeine on growing cells........... 51 Caffeine lowers n2 mRNA levels.................... 53 The inhibitory effect of caffeine is reversible... 55 Xanthine inhibits cell growth..................... 57 Caffeine reduced D2 transcript levels but induced 029 gene expression....................... 59 Hydroxyurea does not affect D2 mRNA levels........ 59 starvation or cell-cell contacts are not sufficient signals to lower D2 mRNA levels........ 63 Non-oscillating extracellular cAMP levels do not affect 02 gene expression in XPSS strain...... 67 (Fig. A, B, c and n). Elevating the intracellular cAnP levels do not affect the D2 gene expression................. 70 Oscillation of extracellular cAuP levels is necessary to lower the D2 mRNA levels.......... 73 ix IV I‘I 7B. 10. 11. Page Polio acid pulses do not affect the D2 transcriptlevels................................ 78 The expression pattern during suspension development with the addition of lug/ml amino acids, lmu folic acid or cycloheximide. . . . . 80 Activation and deactivation of a cell surface chP receptor is necessary for lowering the 02 .mlavel‘OOOOOOOOOOOOOOOOOOOOO0.0...0.00.0.0... 83 signal transduction mutants show a defective response to can? pul'......OCCOOOOOOOOOOOOOOIO... 87 starvation inactivates the 02 gene transcription but activates the transcription of the D29 gene.. 91 12. A model for the regulation of D2 gene expression. 124 INTROWCTION In order to study the regulation of gene expression, it is important to have model systems. WM discoideum, is one such favorable system for studies on the regulation of growth cell-specific gene expression using available molecular biology and molecular genetic techniques. The organism has a small haploid genome and exhibits an experimentally useful transition. from proliferating vegetative cells to non- proliferating differentiating cells. The life cycle D. discoideum is a soil amoebae 'which grows on bacteria as the food supply (Bonner, 1967). Although it is difficult to grow the wild-type isolates in a defined medium in the absence of bacteria, several derivatives of Q. discoideum NC- 4 (i.e Ax-2 and Ax-3) were selected for axenic growth in liquid medium (Ashworth and Watts, 1970; Watts and Ashworth, 1970: Pirtel and Bonner, 1972 aab: Loomis, 1971: Rossomando and Bussman, 1973). Pood exhaustion and starvation for amino acids (Marin, 1976) trigger biochemical and morphological differentiation in Dictyosteligm. Upon starvation, the cells secrete cAMP which orients chemotaxis of the amoebae into multicellular aggregates of 105 cells and these pass through other distinct morphological stages: mound, mound with tip, 2 slug and mature fruiting bodies which contain two cell types, spore and stalk cells (Loomis, 1975) . Under optimal environmental conditions, and with continued starvation, the differentiation process is completed within 24 hours (Fig.1) . These morphological changes are closely linked to specific changes in cellular enzyme and protein composition and correlate with changes in gene expression (Alton and Lodish, 1977: Pirtel, 1972: Gussman and Osmond, 1964 and 1976: Newell and Bussman, 1970). Completion of the differentiation process requires the expression of early and late developmental genes. Genes expressed during the pre- aggregation stage (i.e. discoidin I and cAKP receptor genes) are called early developmental genes, while those expressed after aggregation (i.e. prespore and prestalk genes) are late developmental genes. Another-chemoattractant of Q. discoideum cells is folic acid. It is believed to be used by amoebae to sense the bacterial food sources (Pan gt _a_l., 1972, 1975: De Wit and Bulgakov, l986a,b,c,d). Polio acid is a more effective attractant of preaggregation amoebae, attracting vegetative cells and early developing cells until cAnP signals are initiated (Pan gt _a_]_.., 1972 and 1975). When supplied in pulses, folic acid induces biochemical oscillations accompanied by periodic changes in cAMP concentration and stimulation of the development of preaggregation amoebae to DEVELOPMENTAL CYCLE OF DICTYOSTELIUM DISCOIDEUM 0 «Germination Spore amoeba . Ohrs. Mature ' fruiting body 26 hrs. 24 hrs. . Aggregation / 7-0 hrs. 20'2‘ hfs. g! Aggregaie '2 hrs. Culminoiion / \ . m- 61.7? , " /Migroting Mexican \h pseudoplosmodium IB-IS hrs F IGIRE 1 4 aggregation-competent ones (Wurster and Schubiger, 1977) . If these aggregation competent cells are removed from the suspension culture and transferred onto a coverslip, they immediately aggregate into streams (Gerisch, 1968) . The organisation of the genome L discoideum has a genome sise only about 1% that of mammalian cells. A single haploid cell contains about 4.!5x107 base pairs of DNA and is about seven times the sequence complexity of z. 9&1)“ three times that of yeast, and one sixth that of grosgphila (Pirtel and Bonner, 1972a) . The genome has A-T regions (~ 72% of the genome) and exhibits similar gene organization and transcription as higher eucaryotes. It contains repetitive and single copy sequences interspersed (Pirtel g3; AL, 1976 stub), and the combination of coding, non-coding and untrenscribed (Minty and Newmark, 1980) sequences is- also found (Kimmel and Pirtel, 1983). If all the genome coded for mRNA; there could be up to 20,000 genes. However, about half of the DNA present as A-T rich sequences is not transcribed. RNA hybridization analyses indicate that growing cells express only about 5,000 genes. Nany of these genes are also expressed throughout development (Pirtel, 1972). The genes of Qictyostelium have structural features common to other eucaryotes like the Goldberg-Hogness box (the "TATA" box) and the "CAAT" box, found, respectively, 5 about 31 and 80 nucleotides upstream from the eucaryotic transcription initiation sites (Denoist 23 $1., 1980). These conserved sequence elements (the TATA homology and the CAAT homology) are believed to determine the fidelity and efficiency of transcription in yin and in mm in most eucaryotic systems studied (Ninty and Newmark, 1980: McKnight and Kingsbury, 1982: Breathnach and Chambson, 1981). Unlike other eucaryotes, many but not all, W genes contain an oligo (dT) sequence, 5' to the transcription site. The 5‘ upstream region contains two long homopolymers, respectively, of T and A and the 3‘ flanking regions also contain almost homopolymers of A and T. Dictyostelium has also a consensus sequence AAUAAA following the stop codon. This sequence is a poly-adenylation signal in higher eucaryotes and is found at the 3‘ ends of all the Q. discoideum genes analyzed (Kimmel and Pirtel 1983: Proudfoot and Brownlee 1976) . Dictyostelium mRNA molecules, as in other eucaryotes, contain a 3' terminal polyadenylic acid sequence (Adesnik and Darnell, 1972; Darnell gt 51., 1973) and a 5' terminal cap structure, the unusual nucleotide, 7-methyl guanosine, linked to a 5'-5'-triphosphate bridge (Shatkin, 1976). Both are added post-transcriptionally. Gene expression during growth and development Global analyses of gene expression during growth and development have resulted from solution hybridization 6 analysis of RNA. Growing cells synthesise about 4000-5000 different mRNA-sised, polyadenylated sequences found in the cytoplasm. The same value is obtained from the polysomes which indicates that the corresponding genes (4000-5000) are actively expressed..An additional 9000 transcripts are found in the nucleus, but are transferred very poorly, if at all, to the cytoplasm (Blumberg and Lodish., 1980). During aggregation, abundant RNA species of vegetative cells were found to drop to low levels (Jacquet gt g;., 1981). New species of mRNAs, however, are induced before and after aggregation. During the preculmination stage (~ 18 hour of development), the newly synthesised mRNAs are estimated to be 700-900 species. The number of expressed genes during growth and development (~400 proteins) estimated from two- dimensional (2-D) gel electrophoresis (Alton and Lodish 1977: Cardelli gt g;., 1985) represents a minimum estimate since proteins from mRNAs present at less than 10-20 copies per cell would not be detected. Physiological signals regulate gene expression Gene expression in Dictyostelium is affected by cell associated developmental or environmental stimuli such as starvation, cell-cell contacts, cANP and other factors (Marin, 1976 and 1977; Landfear and Lodish, 1980: Chung gt g_l_., 1981: Nangiarotti gt gt” 1982; Nangiarotti gt g1.” 7 1983: million gt 51., 1984: N011” and .Pittol, 1985). alienation Narin (1976, 1977) showed that the transition from growth to development is initiated by amino acid starvation. Amino acids are the only nutrients that specifically inhibit the initiation of development. There are two classes, essential and conditionally essential. The essential amino acids are: methionine, glycine, leucine, isoleucine, veline, arginine, histidine, tryptophan, phenylalanine, threonine and lysine. These amino acids have .the following properties: (1) each is required to inhibit the initiation of development maximally, even when all other amino acids arecpresent: and (ii) each is specifically required for meximal~growth, even when all other nutrients are present. 21 .gisggigggn is either deficient in the endogenous~synthesis or the retention of these amino acids, therefore, an adequate exogenous source is required to maintain growth. The conditionally essential amino acids (Glutamic acid, Glutamine, Aspartic acid, Asparagine, Alanine, Berine and Praline) have the following properties: (i) individually they are not essential for inhibiting the initiation of development: (ii) individually they are not essential for growth: (iii) as a group, they are essential for the amino acid-mediated inhibition of the initiation of development in the absence of, but not in the presence of, glucose: and (iv) as a group they are essential for growth in the absence of, but not in the presence of, 8 glucose. L gigggtgggg can synthesise the conditionally essential amino acids from Rrebs' cycle and glycolytic precursors. These conditionally essential amino acids required for growth in the absence of glucose can also serve as a source of "Rrebs" cycle and glycolytic intermediates or be utilised as carbon and energy sources for cell metabolism. Without glucose, available pools of the conditionally essential amino acids would be depleted more rapidly than the essential amino acids. The resulting starvation for the conditionally essential amino acids would then initiate development. Adequate endogenous levels of the conditionally essential amino acids are maintained in the cells by the addition of glucose or those amino acids to the medium. Under these conditions, the essential amino acid pools become the limiting factor for starvation. The role of glucose in enhancing the amino acid-induced inhibition of development, or slightly inhibiting when present alone may spare the conditionally essential amino acids. There is no positive evidence to support any direct role of glucose in the primary regulation of the transition from growth to development. Chisholm (1987) found starvation to be sufficient for the induction of cANP receptors whereas cANP pulses might further increase numbers of receptors (Chisholm gt gl. ,1987: Schaap gt gt” 1986). Receptor accumulation during development might therefore be a two-step process. An 9 additional prestarvation response occurs as exponentially growing cells gradually deplete their food supply (Clarke gt 3;. , 1988). Synthesis of certain proteins, discoidin I and the lysosomal ensymes alpha-mannosidase-l and beta- galactosidase-2 (Clarke gt g1., 1987) , was detected approximately three cell generations before the food supply became limiting for growth. nigtygstglim amoebae produce a soluble substance (prestarvation factor, P8!) that accumulates in the medium in direct relation to cell density and can induce these proteins. The cell density required for. induction of these proteins is also affected by the concentration of food bacteria. This cellular response to environmental conditions during growth is distinct from starvation responses and was termed the prestarvation response, to distinguish it from the starvation response that occurs in the absence of bacteria or axenic broth. Cell oontggts Cells; acquire‘oell adhesion surface molecules (contact sites A) which enable them to‘ adhere to other cells during aggregation and in the multicellular aggregates. Evidence has been presented that the: appearance of these contact sites on the cell surface requires exposure to pulses of cANP which the cells normally encounter during chemotactic signaling in aggregation (Gerisch gt gt. , 1975). The immunological studies suggested at least three glycoproteins, with molecular 0g. 10 weights of 80,000, 95,000 and 150,000 daltons ( Parish gt g1., 1978: Steineman and Parish., 1980: Geltosky gt 31., 1976 ), plays some role in developmentally regulated cell-cell adhesion. my studies suggested that the interaction between cell-to-cell surface molecules also controls cell differentiation and the regulation of developmental gene expression. The induction of ~ 2500 new mRNA species (aggregation-stage mRNAs) occurs concomitant with formation of tight-cell-oell aggregates (Nangiarotti gt 31., 1981b: Chung gt 31., 1981). Furthermore, disruption of cell-cell contact results in decay of these mRNAs and in rapid cessation of synthesis of postaggregationrstage proteins. Pinney and his co-wonkers proposed that the 7 hour period between the initiation of development and the onset of multicellular morphogenesis, is. composed of two rate limiting components which are identified by a lack of, or dependency on, cell-cell contactscand fig gm protein synthesis (Pinney gt 91., 1985); The: first component, which includes the initial 4.5 .hours, will progress in the absence of close cell-cell contacts as :well as new protein synthesis. In contrast, the second one that includes the final 2.5 hours depends on cell-cell contacts and protein synthesis. They also tested the developmentally associated changes in protein synthesis during the first and the second rate-limiting period. They found significant increases and decreases in the 1]. rates of protein synthesis of approximately 262 polypeptides. The majority of changes (748) that occur during the first rate-limiting component are independent of close cell-cell contacts, while the majority of changes (66%) accompanying the second period occur in the present of cell-cell contacts. These results might suggest that many genes expressed or repressed during the first 4.5 hours of development do not need cell-cell contacts or protein synthesis for their expression or repression. M Cylic ANP is an important second messenger and it affects many processes in other organisms (Robison gt gl., 1971) Its role in growth gene expression is, however, poorly understood (Paul and Robison, 1984). In 12. gigggiggn‘ cANP serves as a chemotactic agent and also influences the expression of large numbers of genes during early and late development. Aggregation is coordinated through a developmentally regulated cANP signaling system by periodic synthesis and secretion of cANP approximately at 6 minute intervals. , The two responses essential to this coordinated aggregation are chemotaxis (cells move up a cAHP gradient) and signal relay (i.e. the synthesis and secretion of cANP in response to extracellular cANP) . Both are mediated by cell-surface cANP receptors and provide excellent models for transmembrane signaling in eukaryotic cells (Gerisch, 12 1987). van Baastert and others classify cANP receptors into two classes depending on the rate of dissociation of cANP ( Van Haastert, 1987 and Van Haastert gt 31., 1989) . The fast receptor present at ~ 50-100 zloilcell is probably involved in the activation of adenylate cyclase, while the slow receptor (~4n03/c'ell) is associated with the chemotaxis response. Activation of the chemotaxis pathway is accompanied by a 2-3 fold increase in inositol triphosphate (1,4,5-IP3) levels as well as a transient 5-10 fold increase in cGNP levels. The elevation of 1P3 levels might result in an increase in the intracellular Ca2+ levels since the addition of 1P3 to permeabilised cells results in a release of Ca2+ frommmitochondrial stores (Newell gt g;., 1988: van Haastert gt 94., 1989). A cANP receptor gene was cloned by Klein (1988), but it is not known if it represents the cAMP receptor class associated‘with one or*both receptor classes. The two cANP receptor classes could be the products of two distinct genes or different products of one gene resulting in two classes of receptors. Spontaneous oscillations of the fast cANP receptor begin at ~ 3.5 hours of development and continue through 7 hours. The periodic cANP signaling, in turn, regulates early gene expression as well as proper morphogenesis. Oscillation- defective mutants termed "Synag" strains fail to 13 differentiate unless a periodic exogenous cANP is added. However, a constant level of extracellular cAKP, which is necessary for prespore gene expression, does not rescue these mutants, and additionally, suppresses early gene expression in wild-type cells. Oscillations of cAIP'require the surface receptor, adenylate cyclase, and cANP phosphodiesterase. When cANP binds to its receptor, adenylate cyclase is activated and cANP synthesised. If the cells are stimulated with a higher cANP concentration more cANP is synthesised until the receptors are saturated (or desensitised) . After several minutes of continuous occupancy, the receptor 'will be phosphorylated ‘and the cells will adapt and be unable to respond to cANP. Thereafter, cANP-phosphodiesterase will hydrolyze cANP to effect a gradual loss of receptor phosphorylation and the return of the cells to the sensitised state. The cANP ,receptor, identified by photoaffinity labeling (Juliani and Rlein, 1981), oscillates between two interconvertible forms.designated R (40Kb),and D (43 Rb) in parallel with the oscillations in cANP. stimulation with cANP will phosphorylate and convert the R form to the D form. Devreotes and his co-workers propose a model in which there are seven transmembrane domains, the an-terminus is extracellular, and the serine-rich coon-terminal tail is intracellular and is the site of ligand-induced phosphorylation (Klein gt g;., 1988). 14 Another important factor involved in transmembrane signaling are the G proteins. The biochemical and genetic evidence indicates that the cANP-mediated signal transduction pathways are regulated through heterotrimeric G proteins with alpha, beta and game subunits as in mammalian cells (Theibert and Devreotes 1986: Janssens and Van Reastert, 1987: Gnaar- Jagalska gt g1., 1988: Rumagai gt g1., 1989: Pupillo gt g" 1989) . Pirtel and his group discovered and cloned two G-alpha protein subunits, Gel and Gcz (Pupillo gt g1., 1989: kumagai gt g1., 1989). These proteins have ~45% amino acid sequence identity with each other as well as with G alpha protein subunits from yeast and mammalian cells. Furthermore, the GTP/GTPase binding domains show 100% amino acid sequence identity with those of mammalian cells. The Gal protein is expressed in vegetative cells through aggregate stages while Gcz is inducible by cANP pulses and preferentially expressed in aggregation. The Gal appears to function in both the cell cycle and development. overexpression of Gal results in large multinucleated cells that develop abnormally. The expression of Gcz is highly developmentally regulated. Rece88888888888nt results suggest that the Gcz protein subunit is coupled to the chemotaxis receptors that activate phospholipase C that hydrolyzes the membrane bound protein phosphoinositides into 15 inositol triphosphate ( 1,4,5 IP3 ) and diacylglycerol (DAG) (Resbeke gt 31., 1988: 8naar-Jagalska gt 31., 1988: Rumagai gt 31., 1989). The aggregateless mutant strains termed frigid (fgd) strains have been well characterised genetically and biochemically with respect to chemotactic ability ( Coukell gt 31., 1983) . These mutants are unable to respond to exogenous cANP signals. Parasexual genetic studies indicate that the mutants fall in five complementation groups, fgd A, 8, C, D and B. The fgd 8 ,D and D mutants fail to produce detectable levels of cAnP receptors, cANP phosphodiesterase or extracellular phosphodiesterase inhibitor and the cells continue to respond chemotactically to folic acid. Therefore, these strains are probably arrested at the vegetative stage or in early development. strains of groups fgd A and C produce low levels of cANP receptors and secrete phosphodiesterase inhibitor: some of these mutants elicit a weak chemotactic response to cANP. The Prigid A and C mutants initiate development when starved.but the process is blocked at an early stage (Coukell gt 31., 1983). Present data suggest that the Goz protein coupled to the chemotaxis receptor is encoded at the Prigid A locus. strains NCBS and RC112 of the Prigid A group and strain HC317 of Prigid C group were used in our studies. 16 Although it is well known now that the cell surface receptor mediates gene regulation, the second messengers that might be involved in this type of regulation are not clear yet. several studies indicate that intracellular cANP and/or cGNP might not be involved in the gene regulation. For example, although the synthesis of intracellular cANP and cGNP becomes insensitive to continuous stimulation by cANP (Devreotes and stack, 1979) the expression of many late developmental genes requires constant high levels of extracellular cANP (Nehdy gt 31.,1983: Oyama and Blumberg, 1986: Haribabu and.Dottin, 1986: Rimmel, 1987). Furthermore, the proper regulation of late gene expression is still observed in response to exogenous cAMP, despite the inhibition of adenylate cyclase (Rimmel, 1987: Oyama and Blumberg, 1986: Schaap gt 31., 1986). Recent studies show that the addition of IP3 to permeabilised cells results in mobilizing calcium from intracellular stores and in a rapid transient elevation of cGNP (Europe-Pinner and Newell, 1985) . similar responses can be evoked by addition of Ca2+ ions to permeabilised cells. The IP3 and diacylglycerol formed by hydrolysis of phosphoinositides were identified as second messengers in the control of several physiological processes (Berridge, 1984). In _D_._ discoideum, as well as in higher organisms, IP3 stimulates the liberation of Ca2+ from cellular, non-mitochondrial stores, while diacylglycerol l7 activates protein kinase C. To determine whether these second messengers are involved in the cAMP receptor-mediated gene regulation, the effect of various agonists and antagonists that modulate the pathways regulated by 1,4,5 P13 and DAG was studied (Pavlovic gt 31 1988: blumberg gt 31., 1988). They show that the addition of the dihydropyridines, a highly specific class of Ca2 ‘ channel blockers, or Tub-8, a putative inhibitor of calcium release, caused a complete inhibition of cAMP-regulated expression of prespore specific- genes and prevented the repression of a growth gene. Their results strongly suggest that calcium may play a role in the signal transduction pathway. Control of gene expression Gene expression in Digtygstelium, as in other eucaryotes, is controlled at both transcriptional and translational levels. Transcriptional control of eukaryotic genes means that increases or decreases in the synthesis of a primary RNA transcript in the cell nucleus are the main cause of changes in the rate of synthesis of a particular protein. Transcriptional initiation and transcriptional termination are both potentially subject to control, but control of initiation appears to be most important (Platt, 1986). Post transcriptional control can occur at different levels: RNA processing, RNA transport and message stability. Messenger 18 RNA decay (message stability) is now recognised as a major control point in the regulation of gene expression. The changes in the turnover rates of various mRNAs are important to the ability of a cell to replicate and differentiate normally and to respond quickly to changes in its environment. The wide diversity in mRNA decay rates seen both in procaryotes and eucaryotes is evidently due to recognition of some structural features in individual mRNAs. special structures at the 3' termini of’many’mRNAs appear to provide protection against rapid exonucleolytic digestion (Drawerman, 1987), but the selectivity of the decay process appears to be determined to a large extent by interaction between endonucleases or other factors and.internal.mRNA structures. The regulated decay of histone mRNA in mammalian cells requires the presence of a stem-loop structure at the 3‘ end (Levine gt 31., 1987). Bistone mRNA stability probably is achieved by a factor that protects this 3‘ structure from nucleases. Transferrin receptor mRNA also is predicted to form a stem-loop structure which is iron-responsive: the mRNA decays in the presence of iron (Mullner and Ruhn., 1988). This particular sequence, inserted at the 5‘ non-coding region in another mRNA, promotes translation of the mRNA in the presence of iron (Casey gt 31., 1988). Thus, probably there is an iron dependent factor that can control either the translation initiation or mRNA decay depending on the location of this recognition sequence on the mRNA molecule. 19 Furthermore, it was shown that there is a group of AU-rich sequences in the 3\non-coding region in many mRNAs that have short half-lives (Drawerman, 1987) . The. AU-rich sequence probably causes destabilisation of the poly (A) tail whereas a short poly (A) tail often results in accelerated mRNA decay (Wilson and Treisman, 1988) . Nowever, the decay of some mRNAs seems to require more than one structural element: for example the decay of c-‘fos mRNA is determined by the AU-rich recognition sequence as well as another sequence in its coding region (Rabnick and Housman, 1988). Translation of mRNA plays an important role in controlling the degradation of some mRNAs as well. The addition of the cycloheximide, an inhibitor of polypeptide chain elongation, causes stabilization of many unstable mRNAs. Several lines of evidence indicate that mRNA degradation requires the progression of the ribosomes through at least part of the coding region (Graves gt 31., 1987). Per example, it was found that the signal for the degradation of tubulin mRNA lies in the first four amino acids of the growing polypeptide , chain. Therefore, the translation of a portion of the coding region of that mRNA is necessary for its degradation. Although different decay processes were identified, still little is known about how the cellular factors affect the susceptibility of mRNA to degradation. xx. 20 The analysis of mRNA stability in growing and developing nigtyggtgunl cells has received considerable attention as well (Casey gt 31., 1983., Chung gt 31., 1981). The effects of several features of mRNA structure (i.e. 5‘ caps, 3‘ poly (A) tails, mRNA sise and specific sequences within the transcript) in determining mRNA stability were examined (Nanrow and Jacobson, 1988: Nanrow gt 31., 1988). There is a good agreement .that an intact 5‘ cap is essential for the stability of mRNAs, but the destabilisation varies significantly between different mRNAs ( Dru-mend gt 31., 1985: Puruichi gt 31., 1977: Green gt 31., 1983: kreig and Melton 1984: 1978: NcCrae gt 31., 1981) . studies of mRNA with coding region .deletions. indicate that there is no correlation between the size of an individual mRNA and its stability ( Gay gt 31., 1987: Graves gt 31., 1987). The hypothesis that poly (A) tail lengths determine the mRNA stabilities was suggested by microinj ection experiments using adenylated and deadenylated mRNAs (Drummond gt 31., 1985: Hues gt 31., 1974: Marbaix gt 31., 1975: Nudel gt 31., 1976). However, similar experiments with different poly (A)* and poly (A)' mRNAs do not support this hypothesis (Deshpande gt 31., 1979: Hues gt 31., 1983: NcCrae gt 31.,1981: 8ehgal gt 31., 1978). Recent data suggest that there is no direct correlation between the mRNA stability and the length of the poly (A) tail in Q. discoideum (8hapiro _e_t 31., 1988). Furthermore, there is 21 strong evidence that UT rich regions located at the non- coding 5‘ or 3‘ end of mRNAs might play important roles in mRNA stability (Ross and Pizarro, 1983) . Recent experiments show that the deletion or replacement of these UT rich regions results in substantial changes in the decay rate of individual mRNAs (Graves gt 31., 1987: Luscher gt 31., 1985: Morris gt 31., 1986: Rabbitts gt 31.,1985: Shaw and Ramon, 1986: Gimcox gt 31., 1985). The UT rich sequence is particularly important because of the extremely high A-I-T content of noncoding regions in n. gigggiggug mRNAs (Romans and Pirtel, 1985). OBJECTIVES AND ORGANIZATION OF THE THESIS Cell proliferation is a complex process that requires many genes, however, few of those genes have been identified. In eucaryotes, even a good genetic estimate of the number of genes involved in proliferation is unavailable. Because of the unusual life cycle of Q. discoideum some genes whose expression is proliferation-specific were isolated (Ropachik gt 31., 1985). A cDNA library made to vegetative cell transcripts was constructed in the mammalian expression vector pc-D developed by Okayama and Berg (Okayama and Berg, 1982). A subset of 950 clones of the library were differentially screened with 32p-labeled cDNA probes made to vegetative and slug stage cell transcripts. The results show 22 that most of these cloned sequences (646 clones ) represent a class of "vital " genes. These are probably genes encoding gene products necessary for basic cellular metabolic processes or cell structure and the cDNA, therefore, hybridized to both probes. Another class representing 304 genes are termed "proliferative" because they are preferentially expressed in vegetative and cells early in development. We chose ten of those clones that gave the strongest signals of expression in the vegetative cells. These'vegetative‘genes exhibit coordinate regulation: steady state mRNA levels as measured by northern blot analysis show a drastic decrease during aggregation beginning 4 hours into the developmental phase (Ropachik gt 31., 1985). The major objective in this project was to determine the nature of the mechanism regulating proliferation-specific gene expression. The hypothesis that differentiating cells have the ability to repress specifically expression of vegetative genes was examined. At present there is a limited understanding of genetic and physiological signals responsible for the regulation of proliferation-specific gene expression. Whereas little is known about the physiological signals triggering the repression of vegetative gene expression during early development, the induction of differentiation.-specific gene expression is mediated by a variety of signals such as 23 starvation for amino acids, cell to cell contacts, cAMP, differentiation inducing factor (DIP), and other low molecular weight factors (Mehdy and Pirtel, 1985) . These signals might also affect the expression of vegetative genes. In this work, an examination was made on the effect of these signals on the expression of two genes: one is a gene highly expressed in vegetative amoebae and‘whose corresponding cDNA sequence was cloned in the insert of the plasmid pcD-D2 and the other is a differentiation-cell-specific gene, pcD-D29, whose expression begins in the life cycle when D2 gene expression begins to be inactivated. Therefore, the analysis of the D29 gene expression provides a useful comparison. The specific objectives of the project were: 1. To determine the role of starvation in the repression of the D2 gene during development. 2. To determine the role of cell-cell contacts in the inactivation of the D2 gene during development. 3. To test the hypothesis that cAMP might play a role in regulating the selective expression of this gene. since a current hypothesis implicates the involvement of cAMP in the induction and stabilization of some differentiation specific genes. lutants strains defective in the cAMP chemotaxis will be used comparatively. A 4. To determine if the physiological signal(s) affect the D2 gene expression at the transcriptional or post- transcriptional levels. MATERIALS AND METHODS strains I. mc-4 Digtmtgum gigggiggtn NC-4 is a wild type strain from which all the following strains were derived (Raper, 1935): 1. XPSS and ms IP55 is developmentally competent which carries cycloheximide resistance. IP95 is a thermosensitive mutant that grows at 22 °C but the growth is arrested at 27°‘°'C. (Ratner and Newell, 1978). 2. KAI-3 This strain carries the genetic markers axe A and axe B. It was selected for growth in liquid HL5 medium in the absence of bacteria (boomis, 1971 and Bell gt_31., 1976). 3. aces, aciiz and nosiw “‘ These are aggregation deficient mutants termed "frigid" (fgd) strains: they were characterized genetically and biochemically with respect to chemotactic ability (Coukell gt al., 1983: Resbeke gt g1” 1988). The strains are chemotactically unable to respond to exogenous cAMP signals. The HC85 and HC112 strains were derived from the parental strains HC6 and HC91, while HC317 was derived from the XP55 strain. 24 25 Parasexual genetic techniques indicate that HC85 and HC112 strains fall in the complementation group fgd-A, and the HC317 in the complementation group fgd-C. strains of both groups produce low levels of cANP receptors but only HC317 shows some chemotactic response to cANP. The fgd strains appear to initiate development when starved but become blocked at an early stage. II. V121l2 This is a developmentally competent strain (Ropachikzgt 31., 1983) . Growth conditions All the Qt_gtggg1gggg strains, except for KAI-3, were grown in association with 31gtg1g113 pggggggtgg either on 1/2 an agar plates ( per liter: Bacto peptone 5 g, yeast extract 0.5 g, glucose ‘5 g, Mgso4.7 H20 1g, RHZPO4 2.2 g, NaZHPO4 1 g and agar 20 g: Sussman, 1966) or on a suspension culture shaken at 150 rpm. The concentration of amoebae was determined using a hemocytometer and 2x106 cells were dispensed on plates or 5x105 cells/ml in a suspension with pregrown bacteria. The cells were incubated at 22°C. The plates were harvested within ~ 40 hours when the amoebae started to run out of bacteria: or when the cells growing in suspension reached the concentration of 51106 cells/ml. Cells multiply with a generation time of 4-5 hours. A new amoebae culture from spores stored on silica gel was started every month. 26 For axenic strains “-3 and RAT-3 , the spores were. inoculated in ~ 2ml of HL5 medium ( per liter: Thiotone 14 g, yeast extract 7 g, glucose 14 g, Na2P04 0.5 g and kHzP04 0.5 9: pH 6.5) at the concentration of 1x106 spores/ml until they germinated. This culture was used to inoculate 50 ml of HL5 medium in a 500 ml flask which was shaken at 150 rev./minute at 22°C. When the cell concentration reached about 52106 cells/ml, it was diluted into fresh medium at the concentration of 5x105 cells/ml. The axenic cells multiply with a doubling time of 10-12 hours. Developmental conditions The growing cells were harvested when the plates start to clear in NC-4 and HP55 strains or when the axenically growing cells reached the density of 2-31106 cells/ml. The bacteria cells were removed from amoebae by differential centrifugation in the buffer Hz at 1000 rpm: at that speed the amoebae will pellet out while bacteria stay in suspension. The amoebae were washed 3 times in cold xxz buffer(16.6 arr-105204, 3.8 mM-RZHPO” 2 nn-ngso4: pH ~ 6.1). Por normal development, about 21:108 cells were placed on KHZ agar plates and incubated at 22°C. The cells aggregate within 5 hours and took about 24 hours to form fruiting bodies. For suspension development, the cells were suspended at the 7 concentration of 2x10 cells/ml in development buffer (DB) 27 (SmM sazsrou sax NaH2P04, 2.x 119804, :00 mt CaClz: min and Rothman, 1980) . The total volume of the medium was 1/10 of the flask volume. The cells were shaken at.230 rpm to prevent cell-to-cell contacts or at 70 rpm to allow contacts (Nehdy gt 31. , 1983) . Pulsing with can Cells developed in suspension were given cAMP at a 50 nM final concentration at 10 minute intervals. The pulsing began at 3 or 4 hours of development and continued until 12 hours of development. Plasmid preparation Large scale plasmid preparation Asulifimstieamfmm 1. A single bacterial colony was picked from an L-agar plate (per liter: bacto-tryptone 10 g, Bacto-yeast extract 5 g, NaCl 10 g and 2% agar: medium pH is 7.5) containing ampicillin (50 ug/ml) and inoculated in 5 ml of h-broth with ampicillin allowed to grow overnight at 37°C. 2. The culture was transferred to 1 liter of L-broth containing ampicillin and grown at 37°C until the optical density 595 nm (OD595) reached 0.5-0.7 which is the mid-log phase (about 5x107 cells/ml). 3. Chloramphenicol (375 mg) was added to amplify the plasmid. The cleared lysis 28 1. The cells were spun down in GGA rotor at 5,000 rpm for 5 minutes and the cell pellet resuspended in 50 mN tris buffer pH 7.4. Then the suspension was spun again. 2. The pellet was resuspended in 15 ml of 20% sucrose] 50 mM tris pH 7.4 then 1 ml of 15mg/ml lysosyme was added. 3. Three ml of 250 mM HDTA pH 8 was added and the tube was placed an ice for 30 minutes. 4. Twelve ml of Triton.x-100 solution (0.4% Triton 1-100, 50 ml! tris pH 8, 25 mM HDTA) was added slowly while stirring and the mixture was left at room temperature for ~ 10 minutes until it was clear and viscous. 5. The tube was centrifuged at 18,000 rpm in a 88-34 rotor for 45 minutes at 4°C with no brake or was spun in an ultracentrifuge in 30 ml poly-carbonate tubes in a T-865 rotor at 20,000 for 30 minutes. W 1. The supernatant was decanted into a 50 ml centrifuge tube. The chromosomal DNA was strained out with a tea strainer or mesh. 2. The volume was measured and 1 g. CsCl per ml of supernatant was added and dissolved. 3. For every 1 ml of the supernatant, 0.1 ml of 5 mg/ml ethidium bromide (EtBr) solution was added. 4. The supernatant was poured into a 35 ml polyallomer tube and the tube was balanced within 0.1 g with mineral oil or g 29 a CsCl solution of the same density to fill up any extra volume. 5. The tubes were centrifuged at 40,000 rpm for at least 36 hours at 20°C. 6. Two bands were evident under UV light (366 mm, long wave length): the lower hand. contains the plasmid DNA and the upper one is chromosomal DNA. 7 . The tube cap was removed and tube was punctured just below the plasmid to withdraw with. an 18 gauge needle and a 1 ml syringe. 8. The star was removed with salt saturated isopropanol (250 ml of isopropanol with 250 ‘ml of 5N Neel/10 mM tris pH 8.5/1mN HDTA to saturate). One voltuae of saturated isopropanol was added to the plasmid solution (invert to mix), then the tube was undisturbed for ~ 2 minutes to allow phase separation. The Bthr layer was removed and the extraction was repeated until no more star was visible in the CsCl plasmid solution. 1. The plasmid containing solution was poured into prepared dialysis tubing and dialysed overnight against cold 10 all tris pH 8/1mN BDTA (~ 4 liters). 2. The contents of the bag were removed to 30 ml Corex tube, then 1/10 volume of 3 M NaAoc and 2 volumes of cold Ethanol 30 was added. The tube contents were mixed well and stored at 20°C overnight or at -70°C for at least 1 hour. 3. The tube was spun down in HB-4 rotor at. 10,000 rpm for 20 minutes at 4°C to pellet out the plasmid, then the pellet was dried under the vacuum. 4. The plasmid DNA was resuspended in a small volume (200 ul to 1 ml) of TB buffer (:0 mM tris pH 8/1mM BDTA). 5 ul of DNA was placed in 1 ml of H20: the OD260 was measured to determine the concentration (1.0‘ 0D unit a 50 ug/ml) . small scale plasmid preparation MW 1. Bacterial cells were grown in 100 ml L-broth .with 50 ug/ml ampicillin. 2. The cells were spun down in the HB-4 rotor at 7,000 rpm for 5 minutes and resuspended in 2 ml of cold sucrose buffer (25% sucrose, 50mM tris pH 8 and 10 mM BDTA). 3. To the cell suspension, 0.6 ml of lysosyme was added (Sag/ml lysozyme in 50 ml tris pH8/ 10ml! BDTA buffer) and the contents were placed on ice for 5 minutes. Then 1.2 ml of 50m tris/10ml! BDTA buffer and 25 ul of 10 mg/ml RNase were added and the tube was returned to ice temperature for another 5 minutes. 4. Five ml of Triton x—100 solution (50 mM tris pH 8, 10 an BDTA and 2% Triton x-100) was added slowly and the contents 31 were mixed by tipping. 5. To separate supernatant from cell debris, the tube was spun in an HB-4 rotor at 12,000 rpm at 4°c for 10 minutes. 6. The supernatant was diluted to 10 ml with H20 containing 0.4 ml of 5 mg/ml BtBr, then 9.6 g CsCl was dissolved and the tube was spun at 2,500 rpm for 10 minutes to remove the protein particles that were formed as a thin film on the top of the supernatant. 7. The supernatant was transferred to 15 ml polyallomer tubes and the tubes were spun in VT-865 rotor at 50,000 rpm at 20°C for 18 hours. 8. The DNA plasmid was collected, dialysed, precipitated and the concentration was determined as mentioned before. Isolation of D2 and D29 DNA inserts from an agarose gel 1. The cDNA inserts of the D2 gene (0.45 kb.) and the D29 gene (0.4 kb.) cloned in the pcD-Vl plasmid (Pig. 2) were released by cutting the plasmid with BamHl restriction enzyme (~4 units of the enzyme/ug of the plasmid DNA). The digestion reaction was run overnight at 37°C to make sure that the cutting of the plasmid was complete, then the reaction was inactivated by incubating the tubes at 70°C for 10 minutes. 2. The digested pcD-D2 or pcD-D29 plasmid was run on a 1.2% agarose gel for about 3 hours, along with the molecular weight markers, to separate the insert. The gel was stained 32 with ethidium bromide containing H50 (0.5 ug/ml) for ~ 30 minutes. 3. The band of interest was localized using the long wave length UV lamp (366 nm) to minimise the damage to the DNA. 4. The band was cut with a sharp rasor blade and placed into a dialysis bag filled with 0.5: THE buffer then most of the buffer was removed leaving only ~ 2ml or less and the bag was tied. 5. The bag‘was immersed in a shallow layer of 0.5! TBB in an electrophoresis tank and with a voltage of 100 volt for 30 minutes at which time the current was reversed for ~ 30 seconds to release the DNA from the wall of the dialysis bag. 6. The bag was opened carefully and the buffer surrounding the gel slice was collected. The DNA insert was recovered by ethanol precipitation as mentioned before. 33 FIGURE 2 The cDNA cloning vector. The plasmid has the 8V40 early region promoter upstream of ”the cDNA cloning site and an 8V40 late polyadenylation sequence downstream of the cDNA insert. 34 \f X(oONAJ .. .. .. _ If... m... . hf? . 3... $7,... a 9... . . .J .a a . 12.. .. v... a \... . ... . . .!.l v .¢« L .. Ia .. w 3.. L U .OW LI 9. o... ... n): l . o lw.-. . ‘- M \ .m . MWWV/«W. W99. the.“ m % M .. a. ”W M "mm... Gummy .26.!W y////l A x x HM m w 3. .8 R hr! 088322 cri pdvA 35 RNA isolation Total cellular RNA was isolated according to Chirgwin's method (Chirgwin gt 31., 1979) by centrifugation of cells lysed in guanidinium thiocyanate placed over a cesium chloride cushion, or by using the phenol/chloroform extraction method (Jacobson, 1976) . All glassware were baked at 350°C for at least 3 hours and most of the reagents were treated with the RNase inhibitor, diethyl-pyrocarbonate (dep) . Wherever possible, the solutions were treated with 0.1% diethylpyrocarbonate for at. least 12 hours and then autoclaved for 20 minutes. The guanidinium thiocyanate amethod ., 1. The cells (~ 2k108 cells) were dissolved in 10 m1 of 4N guanidinium] 0.1M 2-mercaptoethanol (pH5) and frozen at -70c. 2. To isolate total RNA, the samples were thawed and spun at 3000 rpm/20 minutes to pellet the insoluble materials. 3. In an RNAse free AH-629 polyallomer tube 2.5 ml of 5.7 N CsCl/0.1 N BDTA (pH5) was added to form a CsCl cushion: the sample was layered over the cushion up to ~ 1 millimeter from the top of the tube using 4M guanidinium to balance if necessary. 4. The tube was centrifuged for 24 hours at 25,000 rpm at 15°c. 36 5. All of the sample was removed by aspiration down to the CsCl layer and the tube walls were washed with 3 ml of .dep'd water. The wash of H20 and most of the CsCl were aspirated so that ~ 1 ml is left in the bottom. The tube was inverted and its bottom was cut with a sharp razor blade. 6. The area around the pellet was washed again with 0.5 ml of dep'd H20 and drained with a pipette. . 7. The pellet was jabbed with a blue tip in order to break it up then 0.5 ml of dep water was‘ added and the RNA pieces were transferred to a microfuge tube. 8. Another 0.5 ml of 'dep H20 was added and RNA was broken to small pieces by pipetting up and ‘down several times then vortexed continuously until the RNA was dissolved completely. 9. The microfuge tube was spun for ~ 5 minutes to pellet the insoluble materials, ‘if any, and the supernatant was transferred to a baked corex tube. To the supernatant was added 2 ml of dep H20, 75 ul of 414 NaCl and 7.5 ml of cold 100% Ethanol. After mixing well the tube was stored at -20°C overnight or at -70°C for at least an hour. 10. The tube was spun at 13,000 in HB-4 rotor/20 minutes at 4°C to pellet RNA. The supernatant was aspirated and the RNA pellet was washed gently with ~ 0.5 cold 80% ethanol to remove NaCl residues. 11. The RNA pellet was dried in a vacuum until the ethanol and the H20 were evaporated. The pellet was dissolved in 37 small volume of depId H20 '(~200 ul). The phenol/chloroform method 1. The cell pellet was suspended in 10 ml .of cold HNR buffer (50ml Hopes, 40ml NgC12 and 20m RC1) and to- this was added, with vortexing, 200 ul 10% EDD, 10* ml phenol saturated with 1M tris pH 7.5, 0.5 ml 411 NaCl and 10 ml chloroform. 2. The tube was centrifuged at 3000 rpm for 10 minutes. 3 . The aqueous phase was removed and extracted alternately with an equal volume of phenol and chloroform until the interphase disappeared. 4. RNA was precipitated by the addition of NaCl to 0.314 and 2 volumes of ethanol to the aqueous phase. 5. The tube was centrifuged at 13 ,000 rpm in an HB-4 rotor for 30 minutes, at 4°C and the RNA was resuspended at a concentration of 1 mg/ml in dep'd H20. Northern blot analysis 1. The denatured RNA was sized-fractionated on 1.3% agarose gels containing formaldehyde (Lehrach gt 31. , 1977) and transferred to Gene Screen (New England Nuclear) according to the manufacturer's directions. 2. The filters were baked at 80°C for 2 hours. 3. The blots were prehybridized in hybridization buffer containing (50% Pormamide, 10% Denhardt's solution 50x, 38 5o mM tris pH 7.5, 1% of ultra-pure SDS, :10% Dextran Sulfate, 1 M macl, 1% denatured Salmon DNA and ~ 4% or less H50) at 42°C overnight. 4. Oligolabeled plasmid probes (~ 51:108 to 1x109 cpm/ug) were hybridized to the blots (21106 cpm/blot) in the hybridization buffer for 2 days at 42°C. 5. The blots were washed twice (for 10 minutes each) in 2x SSC ( 0.3M Nacl/.003 M trisodium citrate) at room temperature, and then in 0.5! SSC with 1.0% SDS for 30 minutes at 65°C and in 0.1: SSC for 30 minutes at room temperature. 6. The blots were visualized by autoradiography at -70°C overnight using intensifying screens. Oligolabeling This technique was used for radiolabeling DNA restriction fragments to high specific activity (Peinberg and Vogelstein, 1983) . The technique is very good for radiolabeling small DNA fragments (less than 500 bp). A brief description of this. technique is as follows: 1. Sixty to 100 ng of D2 or D29 insert DNA was diluted to 11 ul in distilled H20 in a 0.5 ml microfuge tube (if the entire plasmid was used, it was linearized with the appropriate restriction enzyme). 39 2. The tube was boiled for 5 mdnutes and cooled quickly in ice in order to denature DNA (single stranded DNA), then the following reagents were added to the tube: A. 18 ul LS ( L8 is a mixture of 1M Hepes buffer pH 6.6: TM : on at the ratio of 25: 25: 7 respectively: TM contains 250mM tris pH 8, 25 MgClz. and 50mM B-Mercaptoethanol: 01. contains 90 O.D. units/ml Hexamers, 1mM tris pH8 and lmM EDTA pH 8). B. 1 ul of 16 mg/ml BSA. C. 3 ul of 100uM dNTPs minus dCTP. 0. 4 ul of (329) corp (3000 Ci/mmol). E. 1/2 ul of DNA polymerase I, Rlenow fragment (9 units). 3. The tube was incubated at room temperature overnight. 4. The unincorporated 32P was removed using the spun column procedure. 5. A one ml disposable syringe plugged at the bottom with glass wool was used as a column. 6. The column was filled with Sephadex G-50 equilibrated in SDS buffer (100mM NaCl, 10mM tris pH 8, 1mM EDTA and 0.4% SDS), then it was spun at about 1,500 rpm for 5 minutes in a bench centrifuge in order to pack down the Sephadex (the packed column volume was about 0.9 ml). 7. The DNA sample was applied to the column and the column was recentrifuged at the same speed and for the same time as before. The effluent from the column was collected in a 40 decapped eppendorf tube which represents the labeled DNA, while the unincorporated (32P) dNTPs remain in the syringe. 8. The counts per minute (cpm) of the labeled DNA (probe) was measured using a scintillation counter and the probe specific activity was determined. Nuclear transcription assays Nuclear run-on transcription assays ‘were performed essentially as described by Nellen (gt 31., 1987: Landfear gt 31., 1982: Williams gt 31., 1980 ). I. Nuclei isolation 1. Axenic cells were grown in the rich medium (HES) until they reached the concentration of 4x10°rcells/ml. Developing cells were shaken on suspension in DB under thevconditions mentioned before. 2. Upon harvesting (3000 rpm/10 minutes), cells were washed 3 times with RR2 buffer. 3. Cells were resuspended in 1/10 of the original volume of lysis buffer II (lysis buffer I + 10% percoll). 4. Cells were lysed by the addition of NP-40 detergent at the final concentration 1%. 5. Nuclei were collected by centrifugation at 5000-6000 rpm for 5 minutes. 6. The pellet was resuspended in 1/10 of the original volume of lysis buffer II, then unlysed cells were spun down at 1000 41 rpm for 5 minutes. 7. The nuclei were collected again from the supernatant as before, and washed once in 1/20 original volume of lysis buffer I ( 50 mM Hopes-pH 7.5, 40 mM NgClz, 20 mM RC1, 0.15 mM Spermidine, 5% Sucrose, 14 mM Mercaptoethanol and 0.2 mM Phenyl methyl sul fonyl fluoride \PMSP') , then they were precipitated again. 8. As soon as the nuclei were isolated, they are used in the tglgtttg transcription reaction. II. In vitro transcription 1. The reaction was set up as follows: A. 34 ul 1120 B. 20 ul 5! transcription buffer (200 mM Tris pH 7.9, 1.25 M RC1, 50 mM MgC12, 25% glycerol and 0.5 mM DDT) C. 5 ul Of 4TH ATP, 4mN GTP and 4mM CTP. D. 1 ul (30 units/ul) of the RNase inhibitor (RNasin). E. 10 ul (100 uCi) of a fresh 32P UTP (3000 Ci/mmole). P. 20 ul nuclei (from 21107 cells) Incubation was at 23 °C for 30 minutes. 2. The reaction was stopped by the addition of: A. 10 ul 1M tris, pH 8.4. B. 10 111 0.214 EDTA. C. 10 ul 20% SDS. D. so ul 1120). E. 200 ul phenol-chloroform. 42 3. Unincorporated nucleotides were removed by passing the aqueous phase over a Sephadex G-50/SDS spun column. 4 . Equal numbers of incorporated counts from each time point were hybridized to filter-immobilised DNA for 48 hours 42°C as mentioned above. Chemicals Unless otherwise stated all the chemicals used in this work were purchased from Sigma Chemical Company. Adenosine 3':5'- monophosphothioate cyclic Sp-isomer (cAMP—S) was obtained from Boehringer Mannheim Biochemicals. Pormamide and guanidinium thiocyanate was from Pluka. 32P-CTP and 32P-UTP was from New England Nuclear. RESULTS I . The expression of the vegetative specific D2 and the differentiation specific D29 genes in vegetatively growing and differentiating cells The vegetative RP55 cells, a derivative of the wild type strain NC-4, were grown with W113 W on 1/2 8M agar plates (Ray and Trevan, 1981) and were collected for RNA isolation before being starved. Por normal development with cell aggregation, the vegetative amoebae were harvested, washed three times with RR2 buffer, placed on RR2 plates (2x108 cells/ plate) and observed to aggregate in response to cAMP. At the end of the aggregation stage tight aggregates with tips had formed by 12 hours. Total RNA isolated from vegetative cells and developing cells (at 4 hour intervals) was size-fractionated on 1.3% formaldehyde agarose gels and electroblotted onto "Gene Screen" membrane. The blot was hybridized with the oligolabelled pcD-D2 and.pcD-D29 plasmids that have the cDNA inserts for these ‘genes. The D2 and. D29 cDNA inserts hybridized to small transcripts on.northern.blots (Pig. 3A). The ethidium bromide stained blot of (A) is shown in (Pig. 3B) as a control for RNA analysis to indicate that equal 43 44 FIGURE 3 The expression pattern of the growth gene (D2) and the early developmental gene (D29) in the developmentally competent strain XP55 during normal development on agar (A). RNA isolation and northern blot analysis were performed as described in the materials and methods section. The autoradiogram shows that the D2 transcripts are abundant in growing cells and early in non-growing differentiating cells but are lost when cells begin to aggregate. The levels of D29 transcripts, in contrast, rise during this period. The ethidium bromide stained blot of (A) is shown in (B) as a control for RNA analysis to indicate that equal amounts of RNA (10 ug) as judged by the equality of the amount of RNA in the 26S and 17S ribosomal bands were loaded in the lanes. All blots shown in this work were stained with ethidium bromide and observed to have an equal RNA loading by this method. 45 Hours 04:12 175‘ 265— 175— ON AGAR 46 amount of RNA (long) were loaded in the lanes as judged by equality of the amount of RNA in the 26S and 178 ribosomal bands. D2 transcript levels were high in. growing cells but fell dramatically by 8 hours of differentiation. D29 transcript levels, however, were undetectable in growing cells but increased by 4 hours of differentiation (Pig. 3A) . Similar results were obtained with V12M2 and NC-4 strains (data not shown). Thus, D2 gene expression is inactivated while the D29 gene expression is activated. The D2 gene might be an important growth gene because its expression is reduced in the life cycle when cells are no longer proliferating. The D2 transcript levels are high during cell proliferation, decline dramatically to undetectable levels during development when the cells are arrested at the G2 phase ( Weijer gt 31., 1984: Rats and Bourguignon., 1974) and increase again upon the resumption of growth after the germination stage (Ropachik gt 31. , 1985). To investigate if cessation of cell proliferation alone affects D2 transcript levels, we used two mutant strains which are thermosensitive for. growth (tsg) , RP95 and HM27. The RP95 mutant, whose defect maps to tsg A on linkage group IV, and H1427 (unmapped mutant) grow normally at 22°C but cease proliferation at 27°C (Ratner and Newell, 1978: Ropachik gt 31., 1983). RNA isolated after 18 hours of incubation at 47 these temperatures was analysed on a northern blot to determine the D2 and D29 mRNA levels (Pig. 4A). The interruption of the cell proliferation.ceused a loss of D2 transcript levels in both strains whereas D29 transcripts were detected only in the HN27 strain. However, the transcript levels of another gene, pr 326, were unchanged which indicates that the loss of 02 transcripts was specific and it was not due to a heat shock response. In the control experiments, where cells grew at 22°C for 18 hours, the D2 transcript levels were high, while D29 transcript levels were not detected. We also blocked the cell proliferation of an axenically growing strain “-3 with caffeine (Hagmann, 1986) to investigate whether D2 transcript levels can be affected in a strain without a thermosensitive growth mutation. In order to determine the appropriate dose of caffeine that arrests cell proliferation, we grew the cells in the presence of different caffeine doses (i.e. 0.0, 0.625, 1.25, 2.5, 5 and 6.25 mM). The 5 mM dose inhibited cell division completely, the 2.5 mM caffeine,had an intermediate effect and the 1.25 mM dose or less had no effect on the growing cells. Thus, caffeine affects the growing cells in a dose dependent manner (Pig. 4B). These results were confirmed by northern blot analysis of RNA isolated from cells under different concentrations (Pig. 4C) Caffeine has a reversible inhibitory effect on cell growth. When the caffeine arrested 48 cells were washed thoroughly with n2 buffer and resuspended in fresh medium, they recovered from the inhibitory effect of caffeine (Pig. 4 D). Purthermore xanthine, a caffeine precursor, was found to have a similar effect as caffeine (Pig. 4 E). Different strains might vary in their responses to the addition of caffeine. Por example, the RP55 strain is less responsive to the caffeine addition in lowering the D2 transcript levels than AR-3 strain (data not shown). When caffeine (5mM) was added to the growing cells, D2 transcript levels were lowered, but interestingly D29 transcript levels increased as if the early developmental events were induced as occurred in the H1427 strain at 27°C (Pig. 4 P). The increase in D29 transcript levels were comparable to that of normal developing cells. Thus blockage of cell proliferation resulting from caffeine or from incubation of some growth mutants at a restrictive temperature lowers D2 and raises D29 transcript levels. These results suggest that the cell cycle might play a role in the regulation of D2 gene expression. On the other hand, when we added to growing cells the DNA synthesis inhibitor, hydroxyurea (1mM), known to arrest the cell cycle at the S- phase, there was no effect on the D2 transcript levels (Pig. 4G) . 49 FIGURE 4A Blockage of the cell proliferation lowers Dz mRNA levels. The expression pattern in vegetative amoebae of two tsg mutants ( XP95 and H1427) incubated at 21°C and after shifting an identical culture to 27°C for 18 hours. The autoradiograms show that the interruption of the cell cycle caused a loss in the D2 transcript levels whereas D29 transcripts in the HM27 strain were induced. The transcript levels of the control gene, pLR326, were unchanged. 50 NNZT 51 FIGURE 48 Caffeine affects cell proliferation in a dose dependent manner. Caffeine was added to axenically growing cells at different concentrations,0, 1.25, 2.5 and 5 mM. 52 $505 08:. me om . 9 o mu W _ n. O I 0.. 23:8 rs e um: 2.2.8 :5 3 1.? 23:3 :5 REIT .2200 I.... .. om 23:8 22:2 waOwam mwOQ wZ_mmn_=mm_n_xm >mm>00wm wz_mmm<0 57 FIGURE 4E Xanthine inhibits cell growth. Ax-3 cells were grown in the presence of different concentrations of’xanthine, 0, 2.5 and SmM. Xanthine at 5mM is as effective as caffeine at the same concentration. 58 A9505 OEE. Nu m.» o * m: \_0 r0.. 2.5.3 as m 1.? 22232 22 a.“ ...T .228 It. . ow 252.8 22:3 meOawwm mwOQ m2_I._.Z