PLACE N RETURN BOX to mantras chockout from you noord. TO AVOID FINES Mum on or baton duo duo. DATE DUE DATE DUE DATE DUE usu IsAnMinnum mm omnnylnumon Wan-9.1 Molecular Cloning of developmentally regulated genes in Dictyosteli um discoideum By Eek-boon Jho A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1995 ABSTRACT Molecular Cloning of developmentally regulated genes in Dictyostelium discoideum BY Eek-boon Jho Dictyostelium is not only a useful model system for the study of development but also for the cloning and characterization of eukaryotic genes. RNAs from vegetative, and 3 h developing, cells were compared by differential display reverse transcription PCR and EHJ-l was cloned. Besides EHJ-l several other cDNAs (Dblp, Dlta4, DdCBS, Drsp24, and Drl7a) were identified as developmentally regulated. The deduced peptide sequences of these clones have about 40 % to 70 % identity to known genes over their entire length in the GenBank DNA data base. The comparison of deduced peptide sequences from DdCBS, DrpsZ4, and Drpl7a to mammalian and yeast homologs showed higher identity between mammalian and Dictyostelium sequences. These data support the notion that Dictyostelium is more closely related to mammals than is Saccharomyces cerevisiae. The homologs of Dblp and Dlta4 are involved in signal transduction in mammalian cells. A homolog of Dblp in rat, RACKl, has a role in translocation of PKC. In Dictyosteli um, myosin heavy chain kinase (MHCK), a homolog of PKC, is mobilized from the membrane to myosin heavy chain upon CAMP stimulation during chemotactic movement. A role of Dblp in the translocation of MHCK was proposed. Leukotrienes act as chemoattractants or second messengers in inflammation or allergic reactions in mammals. However, the presence of leukotrienes or leukotriene synthesis enzymes had not been reported in lower eukaryotes. The significant homology (4O % identity in amino acid sequences) of Dlta4 to mammalian leukotriene A4 hydrolase and conserved residues for this enzyme activity suggested the presence of leukotriene synthesis enzymes and possibly leukotriene related signal transduction in Dictyosteli um. To determine the role of cloned genes, antisense (for EHJ-l Dblp, Dlta4, and DdCBS) or sense (for EHJ-l) RNA producing DNA constructs were introduced. Although the antisense RNA experiments were uninformative, constitutive overexpression of EH]- 1 mRNA caused retardation of development. It may be that EHJ-l encodes a regulatory protein that controls gene expression in growing cells, overproduction may lead to extended production of vegetative specific genes until late developmental stages causing retardation of development. ACKNOWLEDGMENT I has been strengthened by the guidance of God whenever I had hard time and I dedicate this mere work to Him. I would like to express my most appreciation to Dr. Will Kopachik for his guidance and teaching. Sometimes it was hard to follow his expectations and I was frustrated by his toughness in science. All his efforts, however, make me more confident in my future career. I would like to express special thanks to Dr. R. Neal Band for critical comments during lab meetings and careful reading of my manuscripts. Especially he was a great mental supporter to me while Will was on sabbatical. I really appreciate my committee members, Drs. Ronald J. Patterson and Donna Koslowsky for their support, suggestions and interest in my research. I would like to give my respect to my mother, Ok-sun Lee who has prayed for me throughout her whole life. Without her dream and understanding I was not able to continue this work. I also would like to give special thanks to my father-in-law, Songpong Cho for his support and encouragement. I would like to give special affection to my wife, Jimin Cho who shares every moments during my studies. Her support and understanding makes my like more enjoyable during the course of this work. iv TABLE OF CONTENTS Pages LIST OF FIGURES -_ _- ....................... viii References ................... ........... 27 1. CHAPTER ONE : Cloning of a Dictyostelium discoideum developmentally regulated gene EHJ-l and analysis of overexpression - -- u - - - 35 A. Introduction ...... 36 B. Materials and Methods ........................................................................... 3 7 C. Results _ 39 D.Discussion -- - -- -- _ 57 F. References - -- - - - 64 H. CHAPTER TWO : Cloning and characterization of a Dictyos telium discoideum cDNA encoding a G protein [3 subunit-like protein 67 A. Introduction - -- - --- _ 68 B. Materials and Methods ........................................................................... 69 C. Results and Discussion- - ............ - 70 D. References - -- -- - 83 III. CHAPTER THREE : Evidence for leukotriene-related signal transduction in Dictyostelium discoideum ..................... 8 7 A. Introduction - ............ 87 B. Materials and Methods ........................................................................... 90 C. Results and Discussion ............................................................................. 90 D. References _- _ - . ..... - - - ...................... 101 Discussions--- - ........ - -- ............ 104 References ..... . - _ _--- _ ........ 120 APPENDD( I : Primary sequence and developmental regulation of Dictyostelium discoideum ribosomal protein 824 and L7a mRNA - -1 - - ....... ........... 123 A. Introduction -- - -- - - - - ..... 124 B. Results and Discussion - _ - _ - -- - -- - 124 C. References - -- 136 APPENDD( II : Cloning and characterization of Dictyostelium discoideum cDNA encoding cystathionine 13 -synthase ............ 1 3 9 A. Introduction -- -- 140 B. Experimental and Discussion ................................................................ 140 C. References- - -- - 152 APPENDIX III : MATERIALS AND METHODS ............................................ 1 5 4 Growth and Differentiation of Dictyostelium. ................................... 1 5 4 Differential Display PCR... ........ . 157 Recovery and Reamplification of DNA from Sequencing gel ...... 1 S9 Radioactive Probe Preparation ................................................................ 1 60 Screening of A cDNA library -- - 161 Excision of Plasmids from the A ZAP cDNA Clone ........................... 163 A ZAP cDNA library DNA Isolation ........................................................ 1 64 Anchored PCR - - - -- -_ - - ....... 166 Plasmid Isolation ...... - ......... - .......... 167 L Mini-preparation -- - - 167 A. Alkaline method. .......... - - ........ 167 B. Speedprep ..................... - - - -- -- 168 C. Wizard miniprep.-- u - - - - 169 vi II. large Scale Plasmid Isolation ...................................................... 169 A. Cesium Chloride Method. ........................................................... 1 69 B. Qiagen Mam' Plasmid Method ................................................... 170 Transformation of Dictyostelium. ........................................................ 1 7 1 Genomic DNA isolation ............................................................................. 1 7 3 Bacterial Cell Transformation ............................................................... 1 7 5 Southern Blot- ..... 1 77 RNA Isolation ........... 1 80 Northern Blot 1 86 Sequencing .................... 191 References ...... 1 94 vii LIST OF FIGURES Page INTRODUCTION Fig. 1. Iife cycle of Dictyostelium discoideum. ............................... 3 Fig. 2. Four different stages of D. discor’deum. ................................ 4 Fig. 3 . Developmental changes of CAR mRNAs ................................ 14 Fig. 4. Developmental changes of Ga mRNAs ' 18 Fig. 5. A model for signal transduction in Dictyostelium. .......... 25 1. CHAPTER ONE Fig. 1. Differential Display PCR sequencing gel .............................. 40 Fig. 2. Northern blot analysis with PCR amplified DNA (A) or cloned DNA as a probe (B) ........................................ 42 Fig. 3. DNA and deduced peptide sequence for EHJ-l ................ 45 Fig. 4. Southern blot analysis ................................................................ 47 Fig. 5. Developmental Regulation of EHI-l mRNA ...................... 50 Fig. 6. Construction of transformation vector (pDNeo (EHJ-1)) for overexpression of EHJ-l...... ......... -- 53 Fig. 7. Southern blot analysis for transformants overexpressing EHJ-l mRNA. ........................................................... 5 5 Fig. 8. The development of pDNeo(EHJ-1) transformants ........... 58 Fig. 9. Northern blot analysis of pDNeo(EH]-1) transformants.60 H. CHAPTER TWO Fig. 1. Nucleotide and deduced amino acid sequence of DblpcDNA _ -- 7 1 Fig. 2. Alignment of the deduced amino acid sequences of G p subunit-like protein cDNAs ................................................... 7 3 Fig. 3 . Southern blot hybridization analysis ..................................... 7 7 viii Fig. 4. Northern blot hybridization analysis ....................................... 79 111. CHAPTER THREE Fig. 1. Pathways of arachidonic acid metabolism. ............................ 89 Fig. 2. Nucleotide and deduced amino acid sequence of 111112—14cDNA - - 91 Fig. 3. Alignment of the deduced amino acid sequences of leukouiene A4 hydrolase cDNAs ................................................... 93 Fig. 4. Northern blot analysis for Ddlta4 ................................................ 97 Fig. 5. Southern blot analysis for Ddlta4 ................................................ 99 DISCUSSION Fig. 1. Parsirnony of three difl’erent organisms .................................. 1 10 APPENDIX I 9 Fig. 1. Nucleotide sequence of D. discoideum ribosomal protein DrpsZ4 - -- 125 Fig. 2. Alignment of RPSZ4 sequences .................................................... 128 Fig. 3. Expression of DrpsZ4 transcripts ................................................. 130 Fig. 4. Nucleotide and deduced peptide sequence of D. discoideum ribosomal protein Drpl7a. ....................................... 13 2 Fig. 5 . Alignment of RPL7a sequences .................................................... 134 APPENDIX 11 Fig. 1. Nucleotide sequence of the D. discor’deum DdCBS cDNA and deduced amino acid sequence .................................................... 142 Fig. 2. Alignment of cystathionine fi-synthase sequences ............ 144 Fig. 3. Southern blot analysis of D. discoideum genomic DNA ...... 148 Fig. 4. A. Expression of DdCBS mRNA during development. B. Regulation of different types of RNA by cAMP ............... 150 Introduction Dictyostelium discoideum has been used as a simple model system for developmental studies (Loomis, 1982; Firtel et a1., 1989; Devreotes, 1989). Since the growth and developmental processes occur separately, this system is good for analyses of the role of genes in development. Although Dictyostelium has a simple program its developmental processes are often found in more complex organisms such as the vertebrate embryo. Unlike vertebrate systems, Dictyostelium can be cultured in large amounts and is haploid, thus simplifying genetic and molecular biological analyses (Nellen et a1., 1987; Cubitt et a1., 1992; Kuspa and Loomis, 1994). W D. discoideum are soil living amoebae which ingest bacteria by phagocytosis or, in the case of axenic derivates, take up nutrients by pinocytosis. D. discoideum can double their number in about 3 h in the presence of bacterial food sources or 8-12 h in axenic broth. Although D. discoideum has a true diploid phase, formed from opposite mating types as in yeast, most biological phenomena studied are expressed when the cells are haploid. It has about 40,000 Kb of DNA in the haploid genome on seven chromosomes (Loomis, 1982). The formation of a multicellular organism is initiated when cells are deprived of a food source or certain amino acids (Marin, 1976). The developmental process of Dictyosteli um can be divided into four continuous stages: aggregation, mound formation, slug formation and culmination (Fig. 1 and 2; Cardelli et a1., 1985). Starvation enhances the expression of aggregation stage-specific genes and represses vegetative specific genes (Kimmel, 1987; Mann and Firtel, 1987, 1989). Several h after onset of starvation, some cells synthesize and secrete cAMP and nearby cells chemotactically move up the cAMP gradient. The signal relay leads to concentric or spiral cAMP waves that propagate outward at 6 min interval (Gerisch, 1987). When the early development of Dictyostelium on agar is observed by dark- field microscopy both concentric and spiral waves are visible due to the cAMP signaling and migration. Bands of moving cells in response to cAMP signal appear bright, whereas intervening bands of rounded unresponsive cells are dark. The first cells receive the cAMP signal and migrate for only 2 min toward positive gradient of cAMP and then stop until another cAMP signal comes after 5-7 min. During that time cells outside of these receive the cAMP signal and migrate to the concentric center (Alcantara and Monk, 1974; Gross et a1., 1976). At the end of the aggregation period, the radial rings are transformed to streams of cells in which the elongated cells are attached end to end and migrate more rapidly to form an aggregate. The cellular differentiation begins during the mound stage of development (12h) and a group of cells arises in the tip of developing aggregate (Kimmel and Firtel, 1991; Williams, 1991). The tip is thought to coordinate the differentiation of the remaining cells in the later developmental stages and considered to have an "embryonic organizer" role (Durston and Vork, 1979). If the tips are excised and grafted to other host mounds it can define new axes and Germination O -——> - / spore Argo‘eba manor! Loose Aggregate 8 h Tight aggregate 12 h 14h Fig. 1. Life cycle of Dictyostelium discoideum Fig. 2. Development of D. discoideum on agar plate. 0 h (T0), 12 h (mound, T12), 16 h (slug, T16) and 24 h (fruiting body, T24) on non- nutrient agar. s .. Q o a: h 6 thereby cause the formation of several smaller slugs and fruiting bodies (Raper, 1940). The tip secretes cAMP and maintains the gradient of cAMP in the mound and slug. In the slug the tip also coordinates slug migration and appears to be important for cell differentiation (Schaap and Wang., 1984; Williams et a1., 1989; Traynor et 31., 1992). In the slug stages (16h) three different types of cells are spatially localized. The spatial patterning of different types of cells has been identified by using antibodies against cell-type specific markers or using cell type specific promoters connected to a reporter gene, such as lacZ. The anterior 10 to 15% of slugs are prestalk cells which are precursors of mature fruiting body stalks. The posterior three quarters has prespore cells which will form spores in the fruiting body (Rand and Sussman, 1983; Williams et a1., 1989). In the region of prespore some cells are scattered which are indistinguishable from prestalk cells and these types of cells are called " anterior like cells (ALC)" (Stemfeld and David, 1981; Devine and Loomis, 1985). Recently it is clear the prestalk region has at least two subgroups of cells. Prestalk A cells in the anterior of the slug plus ALCs are distinguished by the expression pattern of ecmA (a gene which is induced by differentiation inducing factor (DIF)) (Kopachik et a1., 1983; Williams et a1., 1987; Jermyn et a1., 1987). The prestalk B cells are localized as a cone-shaped group within the anterior of the prestalk zone and distingushed by the expression pattern of ecmB (Williams et 31., 1987, 1989) The proportion of different cell types are controlled by multiple signaling pathways. High level cAMP maintains prespore specific gene expression and leads to formation of spores in the fruiting body (Kay, 1989). Adenosine antagonizes the action of extracellular cAMP and prevents the formation of multiple tips in the aggregate (Schaap and Wang, 1986). Adenosine also inhibits the expression of prespore genes. Differentiation inducing factor (DIF) is another morphogen in Dictyosteli um and it causes the prestalk-specific gene expression and leads the cell fate to stalk (Kay and Jermyn, 1983; Kopachik et a1., 1983; Kwong and Weeks, 1989). DIF appears to be involved in regulating extracellular cAMP level by stimulation of phosphodiesterase gene expression (Podgorski et a1., 1989; Franke and Kessin, 1992). Ammonia does promote spore cell formation by counteracting the effect of DIF (Williams et a1., 1984; Wang and Schaap. 1989). In the culmination stage (24h), a tube of cellulose is formed in the anterior region of slug. The cells in front of the slug migrate through the cellulose tube, form stalk cells, and die. The prespore cells are pulled toward the upper end of forming stalk and differentiate into spores. Finally a mature fruiting body contains about 100,000 spore cells and the spores are held several millimeters above the substratum by a vacuolated stalk. In the presence of food source the spore cells germinate and repeat their life cycle (Loomis, 1982). El'EEE 'l'El Starvation induces the expression of a class of early genes in development and represses the expression of vegetative-specific genes (Kopachik et a1., 1985; Singleton et a1., 1987, 1988). During growth, cells continuously secrete a factor, PSF (Prestarvation factor), that accumulates in proportion to cell density (Rathi et a1., 1991; Clarke et a1., 1988). PSF or a secreted density sensing factor, conditioned medium factor (CMF), induces the expression of early genes (Mehdy and Firtel, 1985; Gomer and Firtel, 1987). The blocking of CMF expression by antisense RNA inhibits aggregation impling that early gene expression is essential for proper development in Dictyostelium (Jain et a1., 1992). In many cases, however the induction or repression of gene expression is controlled by cAMP. About four h after starvation, cells start to secrete cAMP and the gene products for the aggregation process are induced (Mann and Firtel, 1989; Singleton et a1., 1988). These genes encode cAMP receptors, guanylyl cyclase, adenylyl cyclase, phospolipase C, cAMP phosphodiesterase, adhesive contact sites A (csA) and G protein a2 subunit (Kessin et a1., 1992; Gross, 1994). The activation of adenylyl cyclase increases the intracellular cAMP concentration, mediates actin and myosin mobilization and controls chemotaxis (Newell et a1.,1987). During this aggregation period some pre-stalk-related genes are positively regulated by both nanomolar cAMP pulses or continuous stimulation with micromolar cAMP concentrations. Cysteine protease and other proteins of unknown function belong to this group of genes (Barklis and Lodish, 1983; Mehdy et a1. 1983; Mehdy and Firtel, 1985). Several targeted mutants show abnormal development suggesting that aggregation—specific genes are necessary for normal development. 9 After formation of aggregates, the expression of aggregation- specific genes is reduced. During these stages, spore-specific genes are expressed. Micromolar levels of cAMP is required for spore- specific gene expression. The expression of some prestalk-specific genes, such as ecmA and ecmB which encode extracellular stalk matrix proteins, are induced by DIF in this slug stage (Jermyn et a1., 1987; Williams et a1., 1987). In Dictyostelium the expression of many genes are controlled at the transcriptional level. Several cis-acting elements responsible for gene induction by extracellular cAMP, folate, or DIF have been identified (May et a1., 1991; Blusch et a1., 1992; Early and Williams, 1988; Datta and Firtel, 1987. 1988). In case of discoidin 1y, transcription is induced by PSF and repressed by cAMP pulses (Clarke et a1., 1987; Bozzone and Berger, 1987). Sequence elements for both transcriptional induction and repression have been identified by promoter analysis (Vauti et a1., 1990). Most cAMP- inducible promoters have a G/C-rich element in the promoter, termed GBRE (G-box regulatory element) (Datta and Firtel, 1987; Pears and Williams 1987). The removal of GBRE results in a 50 to 100 fold reduction in the level of expression. Firtel's group identified GBRE binding factor (GBF), which is developmentally regulated and inducible by cAMP (Hjorth et a1. 1990; Schnitzler et a1., 1994). They showed GBF is an extracellular cAMP-responsive transcriptional activator which can regulate gene expression. As in other eukaryotes mRNA stability in Dictyostelium is a major control point in the regulation of gene expression (Mullner and Kuhn, 1988; Mangiarotti et a1., 1982; Steel and Jacobson, 1988; Shapiro et 10 a1., 1988). The level of glycoprotein gp80 mRNA accumulates to a maximum level between 4 to 6 h, remains high until 10 h, and then is reduced rapidly to 10% of the maximum level at 12 h (Kraft et a1. 1989). By using in vitro transcription assays, it was found that the rapid reduction of gp80 mRNA level is due to decreased mRNA stability (Chandrasekhar et a1., 1990). Although there are many possible factors determining mRNA stability, no clear cut answer is present. Shapiro et a1. (1988) showed no correlation between mRNA decay rates and the length of poly A tail, the size of mRNA and the number of ribosome per unit of mRNA. They found that unstable mRNAs were more efficiently translated and suggested a translational role for mRNA modifications that change in a time- dependent manner. An unique example for the usage of endogenous antisense RNA in - the stability of mRNA was identified by Hildebrandt and Nellen (1992). The prespore gene, EB4-PSV is constitutively transcribed during growth and development but mRNA levels only accumulate when cells form aggregates. They found that the difference between synthesis and accumulation is due to the developmentally regulated endogenous antisense RNA. During the first hour of development the synthesis of many proteins is rapidly reduced whereas the mRNAs for those proteins persevere in the cell in a translatable form (Alton and Lodish, 1977). It was suggested that a translational control is involved in the reduction of protein synthesis. From the study of distribution of ribosomal mRNAs in polysomes, Steel and Jacobson showed that the blockage of translation initiation is not due to inactivation of these mRNAs by decapping or deadenylation (Steel and Jacobson, 1988). 11 Both papers suggest that lack of soluble factors such as initiation factors leads to rapid reduction in protein synthesis during early development. Post-translational modification is another controling step for the proper gene expression in Dictyostelium. The ribosomal proteins of Dicryostelium are differentially phosphorylated and methylated and those modification are considered an important step for the biosynthesis of the ribosome and (or) its function (Ramagopal, 1990). For proper cell-cell interaction the modification of cell surface glycoprotein is important (Harloff et a1., 1986; Stadler et a1., 1989). Prespore-specific Antigen (PsA) is a 32 KDa glycoprotein isolated from the surface of prespore cells (Gooley et a1., 1992). PsA is post- translationally modified by addition of carbohydrate to the threonine residues of the carboxy-terminal peptide domain, and a glycosyl phosphatidylinositol anchor which attaches glycoprotein to the cell membrane (Gooley et a1., 1992). 5.11 1"12' 1' To coordinate the developmental program, the spontaneous aggregation of thousands of isolated amoebae to a single aggregate, Dictyostelium has to have a well controlled signal transduction mechanism. Konijin et a1. (1968) showed cAMP was an acrasin (a chemoattractant) in Dictyostelium. The binding of cAMP to cell- surface receptors is found in many responses associated with chemotaxis (Dinauer et a1., 1980; Gerish, 1987; Hall et a1., 1989). 12 Induction of developmentally regulated enzyme activity, mRNAs and proteins by addition of exogenous cAMP confirms that cAMP acts as a hormone by directly binding to cell-surface receptor. Four different types (cARl to cAR4) of developmentally regulated cAMP receptors have been cloned. The phenotype of targeted mutants suggests cAMP and cAMP-receptor interaction is essential for development (Saxe et a1., 1991a, b; Johnson et a1., 1993; Saxe et a1., 1993; Louis et a1., 1994). Many recent studies showed heterotrimeric G proteins are coupled to cAMP receptors. Eight different types of Ga and one GB genes have been cloned (Pupillo et a1., 1989; Hadwiger et a1., 1991; Wu and Devreotes, 1991; Wu et a1., 1994; Lilly et a1., 1993). The stimulation of G protein through cAMP bound receptors leads to activation of adenyl cyclase (AC) and phospholiphase C (PLC) (Theibert and Devreotes, 1986; Van Haastert, 1984; Europe-Finner and Newell, 1987). Activation of AC produces cAMP and sends a cAMP signal to outside of cell and causes acitvation of cAMP-dependent protein kinase in the cell. A targeted mutant of adenylyl cyclase A (ACA), which is expressed only in early aggregate, did not aggregate (Pitt et al., 1992). Dictyostelium has a mechanism similar to mammals for phospholipase-mediated signal transduction (Kimmel and Eisen, 1988; Janssens and Van Haastert, 1987; Newell et a1., 1990). Activated PLC synthesizes Ins(1,4,5)P3 (1P3) and diacylglycerol (DG) from phosphoinositol biphosphate. DG and 1P3 regulate gene expression. 13 (A) CAMP receptors. Four cDNAs for different CAMP receptors (CARI-CAR4) have been cloned. They have extracellular amino termini, seven transmembrane domains and long cytoplasmic carboxy-termini (Louis et a1., 1994). CARS share about 60% amino acid identity in transmembrane domains. CARI mRNA is expressed early in development when the CAMP relay system is being established and its level is decreased in late development (Fig.3; Firtel, 1991). As CARI is reduced CAR3 accumulates to a peak at the mound stage and then gradual loss to the fruiting body. The expression of CAR2 is initiated during mound stage and peaks at culmination stage. The cARZ mRNA is enriched in prestalk cells. CAR4 mRNA is initially expressed during tip elongation and continues to accumulate into culmination. The diverse expression of CARs implies that CARs may mediate specific functions at different developmental stages. CARI appears to couple to a Ga2 protein. The addition of guanidine nucleotides to the cell membrane from aggregation competent cells converts the CAMP binding sites from high affinity to low affinity indicating the involvement of G proteins (Janssens et a1., 1986; Van Haastert et a1., 1986). The expression pattern of Ga2 and cARl is parallel. A targeted mutant of CARI by homologous recombination fails to bind or sense CAMP and arrests in early development (Sun and Devereotes, 1991). The expression of CARl utilizes two promoters that are activated at distinct stages of development and respond to different extracellular CAMP conditions 14 N Dd <2 0 3.5 T 3.0 ‘— 2.5 l 1.5 ‘— 1.0 “ 0.0 Loose Agg. Tight Agg. Slug Culml. Fruit. cAMP pulses -- l Preagg. Veg. Continuous cAMP Fig. 3. Developmental Regulation of cAMP Receptors mRNA 15 (Louis et 31., 1993). One promoter is active with low-level oscillation of CAMP; exposure to high CAMP concentrations will repress this promoter and induce a second promoter. CAR2 is structually similar to CARI. Outside of the carboxy terminal region CARI has about 75% sequence identity to CARI in transmembrane domain and loop region. The null mutant of CAR2 shows normal development up to the tight mound stage but arrests development at this stage. This suggests cAR2 may be required for CAMP—directed sorting of prestalk cells during pattern formation within the aggregation mound (Saxe et 31., 1993). The C313“ cell lines display no obvious phenotype (Johnson et 31., I993). The presence of multiple CARs suggests redundancy in cell- Cell signaling strategies in development. The CAR3 (Fig. 3) time course of expression overlaps with CARI and CAR2. The CAR4 mRNA is initially expressed during tip elongation and continues to accumulate into culmination. The car4' cells initially develop normally until aggregation and tip formation (Louis et al., 1994). However, the slugs showed abnormal phenotype in the level of prestalk and prespore gene expression. Certain prestalk markers for prestalk expression is reduced, and prespore genes are expressed in regions normally restricted to prestalk cells. CAR4 may regulate cell type-specific gene expression and pattern formation during the late stages of development. The following table shows the summary of the proposed roles for CARs in Dictyostelium development. 16 Phenotype Change of null cells Proposed role in development CARI Arrest in early development development Fail to sensing CAMP CAMP signaling in early CAR2 Arrest development at mound cAMP-directed sorting stage. prestalk cells of CAR3 role of CAR3 in car3' cells No obvious phenotype changes CARI or CAR2 may substitute the CAR4 Improper gene expression in cell type-specific gene expression slug and culmination in the late stages of development (B) G proteins The Frigid A mutants show no chemotaxis to extracellar CAMP and do not aggregate (Kesbeke et 31., 1988; Mann et 31., 1988). Frigid A cells lack the activation of guanylyl cyclase and adenylyl cyclase and developmentally induced genes are not induced by exogenously applied pulses of CAMP. In severe Frigid A mutants, inhibition of CAMP binding by GTP (a standard indicator of G-protein linked receptors) is not detectable but GTP stimulates wild type level of adenlylyl cyclase activities. These results suggested that Frigid A mutants are defective in a G protein required for proper Dictyos telium development. By using redundant oligonucleotides from the highly conserved sequence in putative guanine nucleotide binding protein of mammalian a subunits, two Ga cDNAs, Gal and Ga2, were initially 17 Cloned (Pupillo et 31., I989; Kumagai et 31., 1989). Six more different Ga cDNAs have been Cloned by PCR (Hadwiger et 31., 1991; Wu and Devereotes, 1991). Each Ga subunit shares approximately 50% sequence identity. Only one GB subunit cDNA has been cloned (Lilly et 31., I993). The comparision of primary sequences indicates they can not be Classified of into any of the Gs, Gi, Gq subtypes in mammals. Ga 1 mRNA is present in vegetative cells through aggregate stages (Fig. 4; Firtel, 1991; ; Wu et 31., 1994). Loss of Gal shows no detectable effects on growth and development (Kumagai et 31., 1991). Possibly other G a subunits substitute for the role of Gal. However the over-expression of G a 1 results in large and multinucleated cells. The majority of cells do not aggregate, and some aggregating cells form small and abnormal fruiting bodies (Kumagai et 31., 1989). G a 1 expression is preferentially expressed in the prestalk AB cells and anterior-like cells. The developmental phenotype of Gal overexpression and cell-type-specific expresison of Ga 1 suggest that Ga I-mediated signalling pathways play an important role in regulating multicellular development by controlling prestalk morphogenesis (Dharmawardhane et 31., 1994). Among eight Ga subunits, Ga2 is the most studied. Ga2 mRNA is induced by CAMP pulses and preferentially expressed in aggregation (Kumagai et 31., 1989). Ga2 null cells do not aggregate and lack CAMP-mediated activation of adenylyl cyclase, guanylyl cyclase and phospholipase C as do Fn’gid A mutants (Kumagai et 31., 1991). G a2 couples to CARI during the aggregation phase of development (Kumagai et 31., 1991). An aa substitution in Ga2, Ga2[G206T], a 18 Cl It... uJaun “ubfi 0301.30:- urn-~— II. u. an; ~M~II OI- I'v- J a is. W“ “Mb-U :0. fig. .DevelopmentalarangesofcamRNAs 19 putative dominant negative mutation, causes an inhibition of receptor-mediated activation of adenylyl cyclase similar to mammalian system (Osawa and Johnson, 1991). Transformed cells with a preaggregation stage-specific promoter controlling expression Ga2[G206T] do not aggregate. However, cells expressing G a2[G206T] under the control of ecmA promoter show normal development through slug formation but have culmination with an aberrant stalk morphogenesis. These results suggest that Ga2 plays an essential role in regulating Stalk morphogenesis as well as early aggregation (Okaichi et 31., 1992; Carrel et 31., 1994). Ga3 mRNA is induced by CAMP pulses and preferentially expressed in preaggregation stage (Fig. 4). The data for null cells of Ga3 is not available. Ga4 is primarily expressed late in development and at a low level during growth and early development. As expected from the expression pattern of Ga4, the Ga4 knock-out cells aggregate and form a tip. However, only the apical portion continues to elongate producing a thin projection that in some cells become "knotted" whereas the basal region remains more rounded (Hadwiger and Firtel, 1992). This mutant forms fewer spores than does the wild type. When ga4' cells co-aggregate with wild type cells normal fruiting bodies. Possibly Ga4 is essential for multicellular development by producing and secreting intercellular signals. GaS is mainly expressed in late development, whereas Ga6 is expressed primarily during growth and very early development (Fig. 4; Hadwiger et 31., 1991; Wu and Devreotes, 1991). 20 Ga7 mRNA reaches a maximum level in preaggregation and followed by lower levels in aggregation and increasing levels until culmination. The Ga7 null cells show no defects in growth and morphology in development (Wu et 31., 1994). Ga8 mRNA level is very low during growth and reaches a maximum level during aggregation followed by declining levels (Wu et 31., 1994). In contrast to mRNA expression the protein is constitutively expressed. like Ga7 null cells, Ga8 null cells do not have detectable phenotypic change. The transformants overexpressing Ga8 do not show any phenotypic Change. These results suggest that Ga7 and Ga8 subunits are functionally redundant with other G a subunits. Only one G13 subunit has been Cloned. The mRNA and protein are constitutively expressed. Its sequence has about 60% identity to the homolog of other systems. It is suggested that this fi-subunit interacts with other eight Ga subunits which are transiently expressed during development. Targeted mutants in G13 subunit are viable, but unable to aggregate (Lilly et 31., 1993). The G 8' cells lack the ability to move towards chemoattractant and their adenylyl cyclase or guanylyl cyclase activity can not be stimulated by CAMP. These results suggest that Gfi links the chemoattractant receptor to effectors and G p is essential in many chemoattractant-mediated processes (Wu et 31., 1995). The following table summarizes the phenotype of null cells and possible roles of G protein subunits. 21 Phenotype Changes of null cells Possible roles Ga 1 No Change Prestalk morphogenesis Ga2 No aggregation; resemble Frigid Link to CARI and signal Amutants transduction in early development and stalk cell morphogenesis Ga4 Normal to mound; abnormal Produce intercellular signals for fruiting bodies development Ga7 No Change May be fuctionally redundant with other subunits. Ga8 No change May be fuctionally redundant with other subunits. Gp No aggregation Not essential for cell viability. Involved in many chemoattractant-mediated processes (C) Effectors for signal transduction Binding of CAMP to surface receptors activates adenylyl cyclase (AC), via a G protein (Kesbeke et 31., 1988; Klein et 31., 1988). AC catalyzes the production of CAMP from ATP and leads to increased intracellular and extracellular CAMP level. An increase of intracellular CAMP leads to activation of CAMP-dependent protein kinase A (PKA). The extracellular CAMP is used for CAMP relay and then destroyed by extracellular phosphodiesterase (PDE). The cells 22 are then ready for another pulse wave of CAMP signalling (Wang et 31., 1988). A group of mutants, Syn3g7, fail to aggregate due to the lack of adenylyl cyclase (Theibert and Devreotes, 1986) Two different types of adenylyl cyclase, ACA (aggregation-specific) and ACG (germination -specific) were Cloned (Pitt et 31., 1992). ACA is expressed during early development whereas ACG is present only during spore germination. CARI and unidentified other receptors are linked to Ga2 and activates ACA activity (Pupillo et 31., 1992). The targeted mutants of ACA are blocked in development and remain as single amoebae. The mutants show chemotaxis to CAMP but do not have adenylyl cyclase activity. Mutants and wild type cells synergize to restore normal development. These results suggest the 3ca'cells can respond to, but cannot produce, a CAMP signal. Moreover CAMP is not required for chemotaxis, growth and cell division which are unaffected in aca'cells. CAMP relay signal activates a phospholipase C (PLC) coupled pathway (Newell et 31., 1988). Stimulation of receptors, through G proteins, activate production of the intracellular messengers diacylglycerol (DG) and 1,4,5 inositol triphosphate (1P3) by PLC. The evidence suggests gene regulation is mediated by the second messenger 1P3 and DG rather than by intracellular CAMP (Ginsburg and Kirnmel, 1989). 1P3 can mobilize intracellular Ca+2 ions which interact with calmodulin (Marshak et 31., 1977) or activate a protein kinase C (PKC). DG binds to PKC and activated PKC may lead to regulation of gene expression. 23 Guanylyl cyclase is another enzyme activated by the CAMP receptor. Evidence suggests the accumulation of CGMP is essential for chemotaxis. First, CGMP accumulation is induced by chemoattractant foliC acid in vegetative, and CAMP in developing, cells (Mato et 31., 1977). Second, the acrasin of Dictyostelium minatum and Dictyosteh‘um Iacteum induces CGMP (De Wit et 31., 1983). Third, mutant stm (Streamer)F remain in the elongated state during chemotaxis for about 5-fold longer than the parental wild type due to lack of CGMP specific phosphodiesterase (Ross and Newell, 1981). Fig. 5 shows a current model for signal transduction in Dictyosteli um. 31.. 1:..“11. Many peOple in Dictyosteh’um research have tried to Clone genes which are induced by CAMP pulse or spore and stalk cell specific genes. However, I have interested in the Cloning of early developmental stage specific genes and the role of these genes during the transition from growth to development. To isolate genes differentially expressed, a method, termed DDRT- PCR (differential display reverse transcription PCR) was designed by Liang and Pardee (1992). Since this method was invented, the DDRT- PCR method has become popular for Cloning of genes (Sager et 31., 1993; Aiello et 31., 1994; Joseph et 31., 1994; Wong and McClelland, 1994; Zimmerman and Schultz, 1994). The DDRT-PCR approach can be used to find many Dictyosteh‘um genes whose mRNAs are induced or reduced by CAMP or otherwise 24 Fig. 5. A model for signal transduction in Dictyostelium. Look at text for details. CAM, calmodulin; DG, diacylglycerol; PIPz, phosphoinositol biphosphate; 1P3, 1,4,5 inositol triphosphate; PLC, phospholipase C; R, CAMP receptors; G2, G a2; G, G proteins; PKC, protein kinase C; PKA, CAMP dependent protein kinase; AC, adenylyl cyclase; GC, guanylyl cyclase; PDE, phosphodiesterase. 25 CAMP ‘1 Outside J R) Pm —> “F N 02 PLC é Inside fg A IP3 Synag 7 5' AMP / ATP AC PDE G CAMP\ Expressior CAMP R m 1...... Differentiation f d 8 Signal Relay CAMP4- R @ d CGMP p Chemotaxis sth fl. GMP 26 regulated. The constitutively expressed genes will be displayed as equal intensity bands. The developmentally regulated genes will be displayed as bands with reduced or induced intensity. I will use DDRT—PCR to Clone induced or repressed genes on early development. After Cloning of genes using DDRT-PCR, effort should be made to determine the role of Cloned genes. The roles of Dictyosteh’um myosin heavy Chain 11 and other genes on development were analyzed using antisense RNA method (Crowley et 31., I985; Knecht and Loomis, 1987; Fang et al., 1993). Antisense RNA producing transformation vectors can be easily introduced into cells to block the expression of targeted genes. Sometimes the transformation vectors are used for the overexpression of genes (Luo et 31., 1994). The following are the main objectives for the thesis. 1. Cloning of developmentally regulated genes by DDRT-PCR. To Clone early induced or repressed genes I will compare RNAs from vegetative and 3 h developing cells. 2. Determine the expression of Cloned genes by Northern blot analysis. DNA fragments from differentially expressed bands on DDRT-PCR will be used as probes. 3. Screening cDNA library and sequencing for cloned genes. To get full size cDNA, cDNA libraries of vegetative, or developing cells will be screened. Sequencing and sequence analysis will be done. 4. Determine the role of CAMP in regulation of Cloned gene expression. Regulation of RNAs during development in shaken 27 suspension cultures with or without CAMP pulses will be Checked using Northern blot analysis. 5. Determine the role of Cloned genes in growth and development by using antisense RNA. The portion of Cloned cDNAs will be inserted into a transformation vector and the growth and development of transformants Checked. If transformants have Phenotypic Changes, Northern blot analysis will be done to Check the level of endogenous RNAs. 6. Overexpression of Cloned genes to determine the role of Cloned genes in development. 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After starvation about 105 cells form a tight aggregate at 12 h, slugs at 16 h and finally a fruiting body at 24 h. Fruiting bodies contain spores which germinate in the presence of a food source (Loomis, 1982). Developmental regulation of gene expression at the transcriptional and post-transcriptional level occurs during the transition from vegetative growing to developmental stages (Mangiarotti et 31., I985; Firtel, 1991; Gross, 1994). Differential screening and subtractive hybridization, have been used to Clone developmentally regulated genes (Sargent, 1987). Liang and Pardee (1992) designed a method, differential display reverse transcription PCR (DDRT-PCR), to Clone differentially expressed eukaryotic mRNA. DDRT-PCR is straightforward and useful under various Circumstances (McClelland et 31., I995). DDRT-PCR was used to Clone highly expressed genes in neonatal mammalian brain and cancer specific genes (Joseph et 31., 1994; Iiang et 31., 1992). D. discoideum is a haploid organism, whose gene expression can be manipulated by transformation (Knecht et 31., 1986). Specific gene expression can be blocked by antisense RNA or homologous recombination (Crowley et 31., 1985; Knecht et 31., 1987; De Lozanne and Spudich, 1987). In addition to blocking experiments, overexpression of truncated cyclin B gene was done to determine the 37 role of cyclin B in Dictyostelium (Luo et 31., 1994). In this Chapter I report the Cloning of a developmentally regulated gene (Clone EHJ-I) by using DDRT-PCR and retardation of development by overexpression of truncated EHJ-l cDNA. MATERIALS AND METHODS Cell growth and development D. discoideum strain KAX4 (a gift from Dr. Rich Kessin, Columbia University) was used for this work. KAX4 was grown in HL—5 (Watts and Ashworth, 1970) in shaking culture (150 rpm) at 220C. Harvested amoebae were allowed to develop on 1.5% Bacto-agar (Difco) with developmental buffer (DB, SmM NazHPO4, SmM NaHzPO4, 2mM MgSO4 and 200uM CaClz, pH6.5). For development in suspension, vegetative cells were harvested and resuspended in DB and shaken at 230 rpm as described by Hassanain and Kopachik (1989). Differential display reverse transcription PCR First strand cDNA was synthesized from 2 p g total RNA of vegetative and 3h developing cells on agar by using SuperScriptTM RNase H‘ reverse transcriptase (Gibco, BRL). PCR (940C, 30 seconds; 420C, 1 minute; 720C, 30 seconds; 30 cycles) was done with arbitrary 10 mer(primer kit, Operon) and anchored poly T (5'- T’TT'TT'TTTTTTT’ITGC-3') primer by using the first strand cDNAs as templates. PCR products were separated on 6% nondenaturing polyacrylamide sequencing gels. A differentially expressed band 38 was cut from the dried sequencing gel and DNA extracted by boiling the gel piece in 150 pl of H20 for 15 min. After separation of insoluble material by centrifugation, DNA was precipitated by addition of linearized polyacrylamide and cold ethanol (Gaillard and Strauss, 1990). Redissolved DNA was used for PCR (94°C, 30 seconds; 40°C, 2 minutes; 72°C, 30 seconds; 40 cycles ) with the same primer pair used for DDRT-PCR. PCR amplified product was separated on 1% agarose gel and a DNA insert was isolated by adsorption to silica gel particles using the QIAEX DNA gel extraction kit (Qiagen). Purified DNA insert was ligated into pCRTMII vector (TA Cloning kit, Invitrogen). Screening of cDNA library and DNA sequencing The insert from the Clone (EHJ-I) in pCRTMII vector was sequenced and used for screening a it ZAP cDNA library made from vegetative cells (gift from Dr. Herbert Ennis, Roche Institute, NJ). The largest Clone from the library screening had 1.8 kb insert. The 11 ZAP Clone containing 1.8 kb insert was isolated as a phagemid by in vivo excision of the Cloning vector as described by Short (Short et al. 1988). The phagemid Clone(pEHJ-1) was subcloned and sequenced using the Sequenase DNA sequencing kit (United States Biochemical). Northern blot analysis Total cellular RNA was isolated by centrifugation of a guanidinium thiocyanate extract through a cesium Chloride cushion or by using RNA isolation kit RNA STAT-60TM (TEL-TEST "B", INC.). 39 Northern blot analysis was done previously described (Kopachik et 31., 1985). Southern blot analysis A 10 u g of KAX4 genomic DNA was digested by restriction enzyme EcoR I, BamH I/EcoR I, Hind III, and Hind III/Xho I and separated on 0.7% agarose gel electrophoresis. DNA was electroblotted onto a GeenScreen (NEN) membrane according to the manufacture's procedures. The blot was prehybridized and hybridized as described previously (Hu et 31., 1992) and washed twice for 30 minutes in 0.5x SSC, 1% SDS at 65°C and then exposed to X-ray film. Transformation of D. discoideum The 1.8 kb insert was ligated into pDNeolI transformation vector to overexpress truncated EHJ-I mRNA constitutively. Twelve ,ug of cesium Chlroride purified pDNeo(EHJ-1) was used to transform exponential phase KAX4 cells by the Ca2+ DNA precipitation method (Nellen et 31., 1987). Stable transformants were selected and grown Clonally in HL-S medium containing 40 ug/ ml of G418. RESULTS Cloning of EHJ-l by DDRT-PCR and screening 2 ZAP cDNA library DDRT-PCR was done with a arbitrary 10 mer(OPA-02; 5'- TGCCGAGCTG) and anchored poly T (NB16; 5'- TFTI'ITTTTITTGC -3') 40 Fig. 1. Differential Display PCR sequencing gel. DDRT-PCR was done with OPA-02/NB16 and OPA-03/NBI6 primer pairs with RNAs from vegetative and 3 h development on agar. PCR products were separated on 6% nondenaturating polyacrylamide sequencing gels. V4 and v5 are vegetative specific bands. ContI shows even expression in two different stages. 41 OPA3 OPAZ 42 Fig. 2. Northern blot analysis. (A) The blots containing 10 [lg of total RNAs from cells of vegetative(V), 3 h and 12 h development on agar was probed with PCR amplified v4 DNA from DDRT-PCR sequencing gel. (B) DNA insert from a Clone, v4-7, was used as a probe. The blot contains 10 pg of » total RNAs of vegetative, 2 h, 6 h and 15 h clevelopment on agar. 43 V T3 T12 -26S -18S V4 V T2 T6 T15 26S - 18S - I”) r—r‘ 44 primer by using first strand CDNA made from vegetative and 3 h developmental stage cells (T3) as templates. A sequencing gel showed two vegetative stage specific bands were in OPA-02 and NB16 primer pair (Fig. 1). With OPA-03 (5'-AGTCAGCCAC-3') and NB 16 primer pair all detectable DNA was evenly amplified between vegetative and T3. Vegetative specific bands v4 and v5 were isolated from sequencing gel and reamplified by PCR with OPA-02 and NB16 primer pair. On agarose gel electrophoresis about 400 bp DNAs were identified. PCR amplified v4 and v5 DNAs were isolated and radiolabeled by the random primer method and then used to probe Northern blot. The signal for v5 was undetctable and for v4 showed two mRNAs whose level is lower in development (Fig. 2A). The two bands identified could be a result of multiple types of amplified DNA in the probe. In order to Clone DNA for both developmentally regulated RNAs, PCR amplified DNA for v4 was Cloned into pCRTMII Cloning vector and inserts were isolated from independent Clones. When Cloned DNA was used as a probe 3 single band was detected (Fig. 2B). A insert from pv4-1 plasmid hybridized to lower band and another insert from pv4-7 bound to upper band (Fig. 2B). The sequencing data for pv4-1 showed 53% sequence identity to mouse 605 ribosomal protein L7a in 136 C-terminal amino acids overlap (Giallongo et 31., 1989). The deduced potential peptide sequences from pv4-7 did not have significant homology to known genes in GenBank DNA data base search. A A ZAP CDNA library made from vegetative cells was screened using insert DNA from pv4-7 as a probe. The largest A ZAP Clone hybridizing to the probe whose 3' end was identical to that of the v4-7 sequence had 45 Fig. 3. DNA and deduced peptide sequence for EHJ-l. A 71 ZAP Clone EHJ-I was sequenced by subcloning or using synthesized primers. Two possible polyadenylation signals were underlined. GenBank accession number is U2 75 40. 46 C’I'ICA'I‘I'ICCI‘CACTACTCATCAAGCCGTAC’ICCICITAAGAGAATTAGAGI‘I‘GTAGGTC SFAHYSSSRTALKRIRVVGQ AAGCATCPATCCCAATCCATCTTPIRGCAAAAGI‘IGATTTAGPPITAGGI‘ITATCAGATT ASMPMHVLAKVDLVLGLSDF TCATICAAGATAACAAAATGAGICAATCAATCAGAGCCACCAAGAAGCAAGITCAAGGTT IEDNKMSESMRATKKQVQGS CAACCG’I'I'ICAA’I‘CACTCCAGCAG'ICATTAAACAATACTA’IGG‘I‘A‘ICCCAACCGGTCAAA TVSITPAVIKQYYGIPTGQI TICGICT'ICAATCAGAAAACPICCAATCAAWCCICCCITUIC‘ICATTICTATTCATCAG GVESENFQSIAAFSDFYSSG WACMWAWWAWTFCATCAACICITAGAGITAAAA ALQFFDQKFGIDSSTVRVKN ATCTIGGTCAAAACICTATTCCCCAAAACICIGATCAAATCGAATCIGATCICGACGWC VGQNCIAQNCDQMESDLDVQ AATATATCACIGCCATCGGTAACAATATCACCACICIUWTCAQZTAATGGTC YMTAIGNNITTLFLSSGNGE AATWATCATICATTGGGCTACIGCCATCCAACAATIACAACCCAATICCAAAGANGCIT WIIDWA'I‘AIQQYNPIPKIAS CAATC’ICATACGGTIGGGCIGAAGTTCAACAATGTGAAATRCAAACAGCICTICTACIC ISYGWAEVEQCEITNSCSTL WATIGAWACWWWGW GIDSVVYVARSNVELQKVGL TACGIGGNWWWTGAWCCAAW RGVSVFVSSGDDGAPSFGAA CCICIUGTAACIGTCCAATUGATGCPACCAAACAA’IRCIGCCCATIAGGKBAWCC SGNCPIDGTKQYCPLGGCNH ATAAATCITICAATC‘ICCAATCATTACCATCATCGAAAGCAACGGPACICAATCITICT KSSQCPMITIMESNGTQCFF TCCCAATCGGITCAGAAAGTRACACPICICAATCTATUPPACAAAACCAAAATATCGICA PMGSESNTCQSMLQNQNIVN ATUGTATCAATGAATPICTIRGCICAAACICTAAATGTCAAGTCGCCCICGAACAAGATA GINEFVSSNSKCQVALEQDT CICAACAAAACTACCACATCTACICTAGCIGTACI‘ICTGACAAATIYIAAACCATACICIC QQNYHIYSSCTCDKLKPYSD ATAWWMQWCWNAMGAWACW SDAGFKIVGYSYDQDAGTLF TCCAACCAGATTATCCAGCITCATCACCATICATCACCICIC‘ITGGICCCACICAAATCA QPDYPASSPFITSVGATQIT CIGATCTTACCAAACCAGAAATIC’ITICTICAGICGCAACICGOGCCATCATTACTGGIG DVTKPEIVCSVATGAIITGG GAGGICGTCTIGCTATCACICAAGCICAACCATCA‘IIACCAAGCIGATGCCGPIGCCACTT GGVAITQAQPSYQADAVATY ACATCAAAAGICGTACIUICCCACCATCA‘IAHCA’HXCATGCCACCAATAGAMTCC IKSGTLPPSYSYMPPIDSIQ AGATCTTACIUPIC‘ITGGTCATGCPFATGAATCGCGIWCAAACACIUICACCI‘CAAATA ILLLLVMLMNRVPNTLTSNT CCICICCATCCGCCITAGAAAGTCTICATCGTACCTCATCTICATCACCAACTC’HCCTC CPCALESVDGTSCSSPTLAG GTATGATCTCITTAATTAATGATAAATTAATICGICCICGIAAACCAACCCI‘CGGITTCT MISLINDKLIGAGKPTLGFL TAAATCCATMTTATDCCAAGCIGCCAAAGAACAACCAAAGETITICAATGATATTACCA NPLLYQAAKEQPNVFNDITT CICGICCAAACAACICTAACAGAGCITACIGTICICAATATGGITACACCGCPACCACIC GANNCNRAYCCQYGYTATTG GTFATCATCCICCCI'CAGGTITAGGITCAATPAACITTAAGAACTITGAACAATACGITT YDAASGLGSINFKNFEQYVL TAACI'I'I‘AAACTAAATAATATAATATAATAAATATATAAA'RAATATATMTCGATITAA SLN CATICAATI'I‘TATTAATAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 47 Fig. 4. Southern blot analysis. 10 [1g of genomic DNA from KAX4 was digested by EcoR I, BamH I/EcoR I, Hind III and Hind III/Xho I and separated on agarose gel and electroblotted. Blot was probed with radiolabeled 1.8 kb insert DNA from the A ZAP Clone EHJ-l. * rel-9““ inf-u- $~ fit”:- I l . I 4‘. L I ‘ ‘e-ax‘: 48 RI B+R HIII H+X Zle - - ~ - ' - 5.0Kb - 4.2kb - 49 1.8 kb insert (EHJ-l). Northern blot analysis determined the EHJ-I mRNA to be 2.3 Kb. The 1.8 kb DNA (EHJ-I) sequence was deposited in GenBank with an accession number U27540 (Fig. 3). The sequence has a conceptual 536 amino acids open reading frame, but may lack about 500 bp more for full size CDNA. The deduced peptide sequence did not have significant homology to other genes in GenBank data base search. To determine the copy number of EHJ-I gene Southern blot analysis was done. A single 6 Kb hybridized band was identified in restriction enzyme cut genomic DNA. Southern blot analysis suggests EHJ-I may be present as a single copy gene in D. discoideum haploid genome (Fig. 4). Developmental regulation of EHJ-I mRNA and down- regulation of EHJ-l mRNA by CAMP pulse Northern blot analysis with the 1.8 kb EHJ-I insert DNA as a probe showed that a single 2.3 kb RNA is hybridized and EHJ-I mRNA level was highest in the actively growing vegetative, but reduced in developmental, stages (Fig. 2B and 5). The expression of some developmentally regulated genes is controlled by CAMP (Mann and Firtel, 1987). To Check whether EHJ- 1 mRNA level was regulated by CAMP, a suspension culture was given CAMP pulses (Hassanain and Kopachik, 1989). Vegetative cells were harvested and resuspended in development buffer (2 x 106/ ml) and shaken for 4 h at 230 rpm. A pulse of 50nM CAMP was given every 10 min between 4 and 8 h (T8P and T12P in Fig. 5). The cyclic AMP pulse concentration and delivery interval replicate the 50 Fig. 5. Regulation of EHJ-I mRNA on development and by CAMP pulse. RNAs were isolated at Vegetative (Ax), 1h (T1), 2h (T2), 6h (T6), 9h (T9), 12h (T12), 15h (T15) and 24h (T24) stages of development on agar. To Check the regulation of EHJ-l mRNA by CAMP pulses RNAs were isolated from cells of shaken suspension culture in DB. Vegetative amoebae were resuspended at 2 x107 cells/ ml in DB buffer. After 4hr some cultures received additions of 50nM CAMP every 10min for the next 8hr. Total RNA was isolated from cells without(Tl ZS) and with CAMP pulses(T12P) in suspension culture. The blot was probed with a radiolabeled 1.8 kb insert DNA from the 4 ZAP Clone EHJ-I. The constant expression of Dblp mRNA was used for even loading control. 51 Ax T1 T2 T6 T9 T12 T15 T24 T88 T8P T128 T12P EHJ-l .---—o- ..... - - Wag... . Dblp 26" J43 Rel. Ell prt PT‘I cut ex; ap; Ira 501 52 physiological rise and fall experienced by aggregating cells as amoebae relay and then degrade a cyclic AMP signal. The EHJ-I mRNA level in CAMP-pulsed cells was about 10 fold less by 4h than control (without cAMP) and undetectable by 8 h (Fig. 5). Suspension cultured cells without CAMP pulse in development buffer had a nearly identical amount of EHJ-I mRNA as do vegetative cells. The hybridization pattern for Clone Dblp shows that an equal amount of RNA was loaded in all lanes. Whether EHJ-I mRNA level is controlled at the transcriptional level or post-transcriptional level is unknown. We conclude that the CAMP relay during normal development on agar plate is essential for down-regulation of EHJ -1 mRNA. Retardation of development by overexpression of truncated EHJ-I mRNA The largest EHJ-I Clone has 1.8 kb insert (Fig. 3). A translation product from EHJ-I may lack the N-terminal region of the EHJ-I protein. A 512 amino acid polypeptide should be synthesized from the 1.8 kb insert in transformation vector pDNeoII. Pst I and Xho I cut 1.8 kb DNA was inserted into pDNeolI and transformed into exponential growth phase KAX4 cells (Fig. 6). Several tiny Colonies appeared after 7 days selection with 40 rig/ml of G418. Three transformants were isolated and two were further analyzed. The Southern blot analysis of two transformants showed the expected 1.3 kb Pst I/Bgl II fragment in both transformants, implying no illegitimate recombination in those transformants (Fig. 7). Both transformants grow at the same rate in axenic medium as control 53 Fig. 6. Construction of transformation vector (pDNeo (EHJ-I)) for overexpression of EHJ-I. The insert of EHJ-I in pBluescript 11 SK (Stratagene) was cut with Pst I/Xho I and ligated into Pst I/Xho I cut pDNeoII vector. K stands for restriction enzyme Kpn 1 sites in EHJ-I. S4 pDNeoII '. . . ', a 7' ' , . ' ' ' . , ' ' ‘ . . . ,* 5 . . .' 3:83.»...-1............._... ' ' ~ - ' . . ’ '- ' .‘ -- ‘ ;-:-:-;-;-:-:-: -; -. ' A8-3' K K K X 0.8Kb Io.3srBm03mOm>H QZZ>M>qxozoum>H AZE>¥>QMDBOUm>H AZB>M>Z¥OZOOm>H HQBHM>MZDZmOm>H 00.0.. o. co. BZZAmHmmomm¢m>H BZBJMHBXDmmOm>H BZBQXHBxDmmOm>H BZBQMHexnmmOm>H BZB>MHBHmeommmB>H QMBZHHfixflmma>mmommqua JOB>Z>BXDmmmmHH 0 000000... 000 Bm.OaZme Hm.meme Hm.meme Hm.ZBBme Bmfizommom omzo>m omzomm omzomm omzo>m omzomm mzoomm mooamm mooamm mooomm mooomm 20m.ZQ>< 20mm.0m8 20mm.0m8 BmemQam BDm.mem O mm>0m>3nmmm00 hm>0m>3mmmmma mm>um>3mmmmmfl mm>0m>3m9m0mm >thHd>mq>oxemo>a m<>mn>oxemo>m m<>ma>oxemo>m m4>ma>oxemo>m m¢>mz>ooemoxm 00000.. 000 o a>>QO>mmOmOBq H>>Dm>mmmmwma H>>Dm>hmmm0ma H>>Do>hmmm0ma H>>Do>hmmmOMd 00.. co. oo o B4H309m4m2 B¢H08>BOZmUMJ B¢HOB>BUZmOMd BBZBmOMQ >MOZmO>J oo 0.0 .0 O eomq>qmmomz OOHBWXUMUJ O>BWMU>OQ O>B>XU>OJ mOHwaoqu Umaemxuqu 0.00. 00 mm¢BBOUMB o O o mmmm>O>OmOQ.MB ZBmD.m9 ¢m0mHO>ZBmQ.mB <¥m<>0r2mmm.mm mmmmq>fi>0m¢x00m2 OO QUHM Hedda m.mHm dfino dang QUHM Heodm m.mfim dano dang <0Hu Hmodm m.mfim mane dflno <6Hu Hedda m.mHm daho dang 75 nemmaom oemmdom memoaom ..WmD mBOHB mBUHB .ammo ..Omm HOB>MHQUQBMUmhd >03>m>AZQBNU¢hQ >03>m>AZDB>O¢hd >43>mHOOQB>OmMQ mxw>mHAOQZ>UooMoomdo nozqz¢ooomoommoodm memm >Mme memm memm >M2mm oOooo AmODmm AmOQmm amwomm quDmm amOQmm 3mqmeowH>mzxo qumeoOddmdxm 343m960ddmdxm 3owd>oQO¢mEZ>>UBmOB>BZQ>OBmOHm >9>BZA>UBEUH$ >B>Bz>wwmm0>q HB>BZ>>OB$DBQ 0 00000 0 :2 m490mmm2mmm0M4Q>MaOO>Hmmmm mBm... ..... H>mOMAmD>HHMOm mBm... ..... H>mOMQmQ>HHMOm mBH... ..... zmmmmAOQ>HmMmm mmxxxmommd>Hm44>oxeq 0 000 WQMXOW< meMUMZ wdmmwmz wquUm< wamxwmm BQmQMUZB ZEMQXUZfl ZEXQKUZd ZZMQXUZB BmZUMmmx «OHM Hmodm m.NHm aflho dang woke Hmodm m.mHm danu dang <0Hu Hmudm m.mfim mane mane 76 the repeats are more similar to each other than to any other repeating units. For example, the third WD-repeat in Dblp has 94% amino acid sequence similarity and 85% identity to the third WD- repeat of human homolog. Only one exception was identified. The fourth WD-repeat in Dblp has the highest homology to the third WD- repeat in rat RACK]. However, the fourth WD-repeat in Dblp shows highest homology to the fourth repeat of Chlamydomonas, and tobacco, G13-like protein. (3) Dblp has very short amino- and carboxy-terminal extentions as do other G13-like proteins. To determine the copy number of the Dblp gene, Southern blot hybridization analysis was done. The autoradiograph from each lane of the blot containing genomic DNA digested with a restriction enzyme had a single band that hybridized to the 32P-labeled 1.0 kb probe DNA (Fig. 3). This evidence suggests there is a single copy of the Dblp gene present in the genome. The regulation of Dblp mRNA level at different developmental stages was assessed by Northern blot analysis. A 1.1 kb of Dblp mRNA was constitutively expressed from the vegetative and developing cells (Fig. 4). The closeness of the mRNA to the cDNA size suggests the cDNA sequence is full length. Ron et al. (1994) have a model for the control of the transition from inactive to active PKC. In the inactive conformation a pseudo- RACK domain of six amino acid sequence in the C2 region on PKC (homologous to a RACKI six amino acid domain) occupies the RACKl binding site and prevents access of substrate to the substrate- binding site. In the active PKC conformation RACKl displaces the pseudo-RACK sequence from the RACK] binding site and allows 77 Fig. 3 Southern blot hybridization analysis of Dblp gene per haploid genome of D. discoideum. long of genomic DNA for D. discoideum was digested by BamH I (lane 1), EcoR I (lane 2), BamH I and EcoR I (lane 3), and Hind III (lane 4). The blots were probed with 3JP- radiolabeled 1.0 kb insert of the A ZAP Clone. Zle - " 5.0m: - ' 4.2Kb - 2.5Kb - 2.0Kb - 1.9Kb - 78 . Ans. 79 Fig. 4 Northern blot hybridization analysis. Expression of Dblp mRNA during vegetative and deve10pment stages on agar plate (5mM NazHPO4, 5mM N3H2P04, 2mM MgSO4 and 20011M CaClz, pH6.5). RNAs were isolated at vegetative (veg), 1h (T1), 2h (T2), 6h (T6), 9h (T9), 12h (T12), 15h (T15) and 24h (T24) stages. long of total RNAs were used for each lane and the blot was probed with 32P-radiolabeled 1.0 kb insert of the A ZAP Clone. 8O Veg T1 T2 T6 T9 T12 T15 T24 8] access to the substrate binding site. In Dictyostelium the myosin heavy chain kinase (MHCK), a member of the protein kinase C family, is translocated to myosin in response to CAMP (Ravid and Spudich, 1992). Translocated MHCK phosphorylates the myosin heavy chain, and leads to inhibition of myosin thick filament formation during chemotaxis (Ravid and Spudich, 1989; Pasternak et al. 1989). It is possible that Dblp plays a role in the translocation of MHCK. A comparison of Dblp and MHCK amino acid sequence by the Bestfit program (Devereux, 1991) found the amino acid sequence VMVWQL (46-51) in Dblp showed strong homology to MHCK sequence VMIWHL (311-316). The homologous sequence falls in the C2 region of MHCK as does the pseudo-RACK sequence of rat PKC (Fig. 5). The single G13 subunit may participate in the formation of heterotrimers with all of the a subunits in Dictyostelium (Wu et al. 1995). The normal growth of Gfi-null cell suggested that heterotrimeric G proteins are not necessary for cell growth (Wu et al. 1995). It is not known if the G13-like proteins form heterotrimers. Therefore it will be important to determine if Dblp can interact with other Ga subunits to assess the role of G protein related signal transduction in cell growth. Disruption of Dblp gene by homologous recombination should enable us to determine the role of the G13 like protein in growth and development of Dictyostelium. Acknowledgments We thank Drs. Neal R. Band and Ronald 1. Patterson for helpful comments on the manuscript and Dr. Herbert L. 82 C 2 region I—'—I 316 3” VMIWHL N-terminal MHCK inactive C—terminal N-terminal C—terminal Dblp 46 51 VMVWQL N-terminal MHCK active Active site is open. Fig. 5. Model of inactive and active forms of MHCK. DG, Diacylglycerol; Ca, Calcium; PS, phosphatidylserine. En 83 Ennis (Roche Institute of Molecular Biology) for providing the vegetative A ZAP CDNA library. References Bimbaumer L (1992) Receptor to effector signaling through G proteins: roles for By dimers as well as 0: subunits. Cell 71: 1069- 1072 Codina J, Yatani A, Grenet D, Brown AM, Bimbaumer L (1987) The a subunit of the GTP binding protein Gk opens atrial potassium channels. Science 236: 442-445 Devereux J (1991) Program manual: Sequence Analysis Software Package, Version 7. Genetics Computer Group, Madison, Wisconsin Gilman AG (1987) G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56: 615-649 Gross JD (1994) Developmental decisions in Dictyostelium discoideum. Microbiol Rev 58: 330-351 Guillemot F, Billault A, Auffray C (1989) Physical linkage of 3 guanine nucleotide-binding protein-related gene to the chicken major histocompatibility complex. Proc Natl Acad Sci USA 86: 4594-4598 Hadwiger JA, Firtel RA (1992) Analysis of Ga, a G protein subunit required for multicellular development in Dictyostelium. Genes Dev 6: 38-49 Hassanain HH, Kopachik W (1989) Regulatory signals affecting a selective loss of mRNA in Dictyostelium discoideum. J.Cell Sci. 94: 501-509 84 Ishida S, Takahashi Y, Nagata T (1993) Isolation of cDNA of an auxin- regulated gene encoding a G protein 13 like protein from tobacco BY-2 cells. Proc Natl Acad Sci USA 90: 11152-11156 Kim D, Lewis DL, Graziadei L, Neer E1, Bar-Sagi D, Clapham DE (1989) G protein 137 subunits activate the cardiac muscarinic K+-channel via phospholipase A2. Nature 337: 557-560 Kumagai A, Hadwiger JA, Pupillo M, Firtel RA (1991) Molecular genetic analysis of two Ga protein subunits in Dictyostelium. J Biol Chem 266: 1220-1228 Kwatra MM, Carson MG, Lefkowitz RJ (1992) Role of 13 7 subunits of G proteins in targeting the fi-adrenergic receptor kinase to membrane-bound receptors. Science 257: 1264-1267 Lilly P, Wu L, Welker D, Devreotes PN (1993) A G-protein 13-subunit is essential for Dictyostelium development. Genes Dev 7: 986-995 Loh EY, Elliott JF, Cwirla S, Lanier LL, Davis MM (1989) Polymerase chain reaction with single—sided specificity: analysis of T cell receptor 5 chain. Science 243: 217-220 Neer EJ (1995) Hetero trimeric G proteins: organizers of transmembrane signals. Cell 80: 249-257 Neer E], Schmidt CJ, Nambudripad R, Smith TF (1994) The ancient regulatory-protein family of WD-repeat proteins. Nature 371: 297-300 Pasternak C, Flicker PF, Ravid S, Spudich 1A (1989) Intermolecular versus intramolecular interactions of Dictyostelium myosin: possible regulation by heavy chain phosphorylation. J Cell Biol 109: 203-210 85 Pitcher 1A, lnglese J, Higgins JB, Arriza JL, Casey P1, Kim C, Benovic JL, Ravid S and Spudich JA (1989) Myosin heavy chain kinase from developed Dictyostelium cells. J Biol Chem 264: 15144-15150 Ravid S, Spudich 1A (1992) Membrane-bound Dictyostelium myosin heavy chain kinase: a developmentally regulated substrate- specific member of the protein kinase C family. Proc Natl Acad Sci USA 89: 5877-5881 Ron D, Chen C, Caldwell J, Jamieson L, Orr E, Mochly-Rosen D (1994) Cloning of an intracellular receptor for protein kinase C: a homolog of the B-subunit of G proteins. Proc Natl Acad Sci USA 91: 839- 843 Schloss IA (1990) A Chlamydomonas gene encodes a G protein 13 subunit-like polypeptide. Mol Gen Genet 221: 443-452 Short JM, Fernandez JM, Sorge JA, William DH (1988) Z. ZAP: a bacteriophage A expression vector with in vivo excision properties. Nucl Acids Res 16: 7583-7600 Simon MI, Strathmann P, Gautam N (1991) Diversity of G proteins in signal transduction. Science 252: 802-808 van der Voorn L, ploegh HL (1992) The WD-40 repeat. FEBS Lett 307: 131-134 Whiteway M, Hougan L, Dignard D, Thomas DY, Bell L, Saari GC, Grant P], O'Hara P, MacKay VL (1989) The STE4 and STE18 genes of yeast encode potential B and 7 subunits of the mating factor receptor- coupled G protein. Cell 56: 467-477 Whiteway M, Hougan L, Thomas DY (1990) Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae. Mol Cell Biol 10: 217-222 Wu 1 86 Wu L, Gaskins C, Zhou K, Firtel RA, Devreotes PN (1994) Cloning and targeted mutations of Ga7 and Ga8, two developmentally regulated G protein a-subunit genes in Dictyostelium. Mol Biol Cell 5: 691-702 Wu L, Valkema R, Van Haastert PJM, Devreotes PN (1995) The protein B subunit is essential for multiple responses to chemoattractants in Dictyostelium. J Cell Biol 129: 1667-1675 87 111. CHAPTER THREE : Evidence for leukotriene-related signal transduction in Dictyostelium discoideum . Introduction A G protein-coupled receptor stimulates phospholipase A2 or the combined action of phospholipase C and a diacylglycerol-lipase, and leads to synthesis of arachidonic acid in mammals (Van den Bosch, 1980; Irvine, 1982). Arachidonic acid can be metabolized in mammals through lipoxygenase and cycloxygenase pathways (Fig. 1; Parker, 1987). The cyclooxygenase pathway leads to formation of prostaglandins. Arachidonic acid is metabolized to 5- hydroperoxytetraenoic acid (5-HPETE) and HPETE is subsequently converted to unstable epoxide intermediate, leukotriene A4 (LTA4) by 5-lipoxygenase. Leukotrienes are a Class of bioactive compounds which have diverse roles in inflammation and hypersensitivity reactions (Sarnuelsson et al., 1987; Samuelsson and Funk, 1989). Arachidonic metabolites including HPETE and leukotrienes have been shown as signal transduction molecules in several different Systems. 1) In human blood polymorphonuclear leukocytes, Occupancy of leukotriene B4 (LTB4) receptors induces the release of lysosomal enzymes, superoxide generation and the chemotactic Inigration and adhesion of leukocytes to endothelial cells (Ford- IIutChison et al., 1980; Samuelsson et al., 1987). 2) In mammalian Systems, G protein coupled activation of LTD4 receptors sequentially 88 increases intracellular Ca++, inositol (1,4,5) triphosphate (1P3), diacylglycerol and then activates protein kinase C (PKC). Activation of PKC increases expression of genes for signal transduction for another cycle of leukotriene synthesis. The newly synthesized leukotrienes are secreted and used for signal transduction molecules (Crooke et 31., 1990). 3) The opening of cardiac muscarinic K+- Channel can be regulated by arachidonic acid metabolites (Kurachi et 31. 1989; Kim et 31., 1989). 4) In the nervous system of the marine mollusc Aplysia, arachidonic acid metabolites are used for second messengers in the direct modulation of K+ Channels (Piomelli et 31. 1987; Buttner et al., 1989). LTA4 is converted into leukotriene B4 (LTB4) by leukotriene A4 hydrolase. LTA4 hydrolase (EC 3.3.2.6) is a cytosoliC enzyme and its CDNA has been Cloned from human, mouse and rat (Funk et al., 1987; Minarni et al., 1987; Medina et al., 1991; Makita et al., 1992). The primary sequence of LTA4 hydrolase has weak homology (about 20%) to aminopeptidases (Malfroy et 31., 1989) and a zinc binding motif Characteristic of aminopeptidase is present. LTA4 hydrolase also possesses peptidase activity (Haeggstrom et al., 1990) Here the partial CDNA sequence of Dictyostelium discoideum LTA4 hydrolase homolog (Ddlta4) and the developmental regulation of this mRNA is examined. Previously the CDNA for LTA4 hydrolase was only Cloned from mammalian system. Therefore this is the first Cloning of LTA4 hydrolase from a non-mammalian cell. 89 Phospholipids Arachidonic Acids W W Prostaglandin IZ-HIlETE S-HPETE '_ ’ 5-HETE IZ-HETE LTA4 LTC4 LTB4’/ Lv 4 i” LTE4 Fig.1. Pathways of arachidonic acid metabolism 90 Material and Methods A A ZAP CDNA Clone (Ddlta4) with a 1.0 kb insert was isolated from a A ZAP CDNA library made from D. discoideum vegetative cells. Its primary sequence was determined by using sequenase DNA sequencing kit (United States Biochemical). RNA isolation and Northern blot analysis were done as previously described (Hassanain and Kopachik 1989). For Southern blot analysis genomic DNA was isolated from D. discoideum strain KAX4 and digested with several different enzymes. The 1.0 kb insert of A ZAP Clone was used to probe the blot. The 1.0 kb DNA sequence encoding Ddlta4 has been deposited in the GenBank/EMBL nucleotide sequence database with accession number U27538. Results and discussion The nucleotide sequence of Ddlta4 contains a single open reading frame (ORF) of 948 bp (Fig. 2). The deduced peptide sequence of Clone Ddlta4 has 59% sequence similarity (39.2% and 36.2% identity) to the carboxy terminal region of the human and mouse leukotriene A4 hydrolase (Fig. 3). Ddlta4 amino acid sequence showed weak homology to rabbit aminopeptidase (27% identity in 167 aa overlap) same as other mammalian LTA4 hydrolases do. Northern blot analysis shows that the full size of mRNA should be 2.2 Kb which is similar to the size of mammalian LTA4 hydrolase mRNA (Fig. 4; Funk et al., 1987). Besides significant homology in size and amino acids 91 Fig. 2. Nucleotide and deduced amino acid sequence of Ddlta4 CDNA. Two putative polyadenylation sequences are underlined. EcoR I and Hind III restriction enzyme sites in this Clone was marked. 92 10 30 SO ECQB I GGCACGAGGCTCATAGTTGGTGTGGTAATTTAGTAACAAATAAATATTGGTCAGAATTCT H E A H S W C G N L V T N K Y W S E F F 70 90 110 TTTTAAATGAAGGTTTTACAGTATTTGTTGAAAGAAAGATTCTTGGTCGTCTTTATGGTG L N E G F T V F V E R K I L G R L Y G E 130 150 170 AAGAAATGTTTGATTTTGAAGCAATGAATGGTTTGAAACATCTTCATGATGATGTTGATT E M F D F E A M N G L K H L H D D V D L 190 210 230 TATTCACACATAAACATCAAGAAGAATTGACAGCATTAATTCCAAATCTTAATGGTATTG F T H K H Q E E L T A L I P N L N G I D 250 270 290 ACCCAGATGATGCATTCTCATCTGTACCATATGAAAAAGGTTTCAATCTCTTATGTTATC P D D A F S S V P Y E K G F N L L C Y L 310 HindIII 350 TTCAATCATTGGTTGGTGTTGCCGATTTTGAAGCTTGGTTAAAATCATACATTTCCAAAT Q S L V G V A D F E A W L K S Y I S K F 370 390 410 TCTCTTATCAAAGTATTGTCGCCACCCAAATGAAAGATTATTTCATTGAATATTTCACAG S Y Q S I V A T Q M K D Y F I E Y F T E 430 450 470 AGAAGGGTAAATCCGAGCAAATCAGTGTTGTAAATTGGAATGATTGGTTCAATAAACCAG K G K S E Q I S V V N W N D W F N K P G 490 510 530 GTATGCCAATTGAACAAGTTGTCTTTGTTTCCCCAGCTGCTAAAGTTGCCAAGGATTTAG M P I E Q V V F V S P A A K V A K D L A 550 S70 S90 CTGAAATCACTTGGATCAAAGATCAAGGTGTCAATGCAACCAAAGATGATATTAAATCAT E I T W I K D Q G V N A T K D D I K S F 610 630 650 TCAAAACTCAACAAATCATTCTCTTTTTGGATACTCTCATTCATTCAACCTCTGAAAAAC K T Q Q I I L F L D T L I H S T S E K P 670 690 710 CATTATCAGTCGATGTTTTAGAGAAAATGGATTCTCTCTATGGTTTCACCGATGTCGTTA L S V D V L E K M D S L Y G F T D V V N 730 750 770 ATAGTGAATACAAATTCAGATGGCAAACATTATGTCTTCACTCTGGTTTAAAGAGAATTG S E Y K F R W Q T L C L H S G L K R I E 790 810 830 AACCAAAAGTTGTTGAATTTTTAATCTCTCAAGGTCGTATGAAATTCGTTAGACCACTCT P K V V E F L I S Q G R M K F V R P L Y 850 870 890 ATCGTGAATTAAATAAGGTTAACCCTGAATTGGCTAAATCCACTTTTAATAAATACAAAT R E L N K V N P E L A K S T F N K Y K S 910 930 950 CTCAATATCATATTATCGCTTCAAAGATGGTTGCAAAAGATTTAGGTTTATAAATGTAAA Q Y H I I A S K M V A K D L G L 970 990 1010 TTAAIAAACTCTTTTTTTTAAAAAAAAAAAAAATAGTAAATATTTCAAIAAAAAAAAAAA 1030 AAAAAAAAAA 93 Fig. 3. Alignment of the deduced amino acid sequences of Dictyostelium and mammalian LTA4 hydrolase cDNAs using pileup program (Devereux, 1991). 0 : conserved sequences in all three Clones. Bold letters : putative zinc-binding site. # : putative active site. 0 : putative proton donor residue. Human Mouse Human Mouse Human Mouse Human Mouse Human Mouse Dd Human Mouse Dd Human Mouse Dd Human Mouse Dd Human Mouse Dd Human Mouse 1 PEIVDTCSLA PEVADTCSLA 51 SLVLDTKDLT SLTLDTKDLT 101 VIEISFETSP VIEISFETSP 151 SVKLTYTAEV SVKLTYTAEV 201 LIALVVGALE LIALVVGALE 251 YVWGQYDLLV YVWGQYDLLV 301 WCGNLVTNKY WTGNLVTNKT WTGNLVTNKT 351 DDVDLFTHKH NSVKTFGETH NTIKTFGESH 401 VAD.FEAWLK GPEIFLGFLK GPEVFLGFLK 451 WFNKPGMPIE WLYSPGLPPI WLYAPGLPPV SPASVCRTKH SPASVCRTQH IEKVVINGQE IEKVVINGQE KSSALQWLTP KSSALQWLTP SVPKELVALM SVPKELVALM SRQIGPRTLV SRQIGPRTLV LPPSFPYGGM LPPSFPYGGM WSEFFLNEGF WDHFWLNEGH WDHFWLNEGH QEELTALIPN P..FTKLVVD P..FTKLVVD SYISKFSYQS AYVEKFSYKS AYVKKFSYQS QVVFVSPAAK KPNYDMTLTN KPNYDVTLTN €34 LHLRCSVDFT LHLRCSVDFA VKYALGERQS VKYTLGESQG EQTSGKEHPY EQTSGKQHPY SAIRDGETPD SAIRDGEAPD WSEKEQVEKS WSEKEQVEKS ENPCLTFVTP ENPCLTFVTP O. C. O O TVFVERKILG TVYLERHICG TVYLERHICG LNGIDPDDAF LTDIDPDVAY LKDVDPDVAY IVATQMKDYF ITTDDWKDFL VTTDDWKSFL VAKDLAE..I ACIALSQRWI ACIALSQRWV RRTLTGTAAL RRTLTGTAAL YKGSPMEISL YKGSPMEISL LFSQCQAIHC LFSQCQAIHC PEDPSRKIYK PEDPSRKIYR AYEFSETESM ANEFSETESM TLLAGDKSLS TLLAGDKSLS RLYGEEMFDF RLFGEKFRHF RLFGEKFRHF O SSVPYEKGFN SSVPYEKGFA SSIPYEKGFA IEYFTEKGKS YSYF..KDKV YSHF..KDKV TWIKDQGVNA TAKEDDLNSF TAKEEDLSSF 50 TVQSQEDNLR TVQSQEENLR 100 PIALSKNQEI PIALSKNQEI 150 RAILPCQDTP RAILPCQDTP 200 FIQKVPIPCY FNQRVPIPCY 250 LKIAEDLGGP LKIAEDLGGP 300 # .0 .0 HE AHS NVIAHEISHS NVIAHEISHS 350 EAMNGLKHLH NALGGWGELQ HALGGWGELQ 400 LLCYLQSLVG LLFYLEQLLG LLFYLEQLLG 450 EQISVVNWND DVLNQVDWNA DLLNQVDWNA 500 TKDDIKSFKT NATDLKDLSS SIADLKDLSS Dd Human Mouse Dd Human Mouse Dd Human Mouse 501 QQIILFLDTL HQLNEFLAQT HQLNEFLAQV 551 HSGLKRIEPK QSKWEDAIPL QSKWEEAIPL 601 QYHIIASKMV SMHPVTAMLV SMHPVTAMLV IHSTSEKPLS LQRA...PLP LQKA...PLP VVEFLISQGR ALKMATEQGR ALKMATEQGR 617 AKDLGL. GKDLKVD GRDLKVD €95 VDVLEKMDSL LGHIKRMQEV LGHIKRMQEV MKFVRPLYRE MKFTRPLFKD MKFTRPLFKD O O O. YGFTDVVNSE YNFNAINNSE YNFNAINNSE LNKVN..PEL LAAFDKSHDQ LAAFDKSHDQ 550 YKFRWQTLCL IRFRWLRLCI IRFRWLRLCI 600 AKSTFNKYKS AVRTYQEHKA AVHTYQEHRA 96 sequence with other homologs, Ddlta4 hydrolase has putative zinC binding sites, an active site and proton donor residue which are conserved in other Clones (Fig. 3; Medina, et al., 1991; Wetterholm et al., 1992). Northern blot analysis indicates that Ddlta4 mRNA levels are developmentally regulated (Fig. 4). Both axenically grown amoebae and vegetative cells grown with bacteria have similar levels of mRNA. Upon starvation the mRNA level is reduced and increased again around 6 h development. The mRNA level shows a peak at 9 h and a steady decrease in later developmental stages. To determine the number of genes for Ddlta4 Southern blot analysis was done with the 1.0 Kb insert probe DNA from the A ZAP CDNA Clone (Fig. 5). Because the A ZAP CDNA done has EcoR I and Hind III restriction enzyme sites, two bands were expected in EcoR I Dr Hind III cut lanes if the gene encoding Ddlta4 is single copy in the D. discoideum haploid genome (Fig. 2). Southern blot data suggests that a single copy is present for the gene encoding Ddlta4 (Fig. 5). The presence of an enzyme for arachidonic acid metabolism suggests several possibilities. First, arachidonic acid metabolites involving signal transduction mechanism may be present in D. discoideum. It is possible that either leukotrienes are new types of acrasin for chemotaxis in D. discoideum or act as second messenger molecules or act in both ways. Second, it is known that LTA4 hydrolase has weak homology to aminopeptidase and has peptidase activity. LTA4 hydrolase may act only as a peptidase in D. discoideum. Third, LTA4 hydrolase has a role in arachidonic acid metabolism and the metabolites have nothing to do with signal 97 Fig. 4. Northern blot hybridization analysis. A) Expression of Ddlta4 mRNA during vegetative and development stages on agar plate (5mM NazHPO4, 5mM NaHzPO4, 2mM MgSO4 and ZOOpM CaClz, pH6.5). RNAs were isolated at vegetative (veg), 1h (T1), 2h (T2), 6h (T6), 9h (T9), 12h (T12), 15h (T15) and 24h (T24) stages. 10pg of total RNAs were used for each lane and the blot was probed with 32P-radiolabeled 1.0 kb insert of the Dlta4 Clone. B) Same blot was probed with 32P-radiolabeled 1.0 kb insert of the Dblp for even loading control. 98 A) veg T1 T2 T6 T9 T12 T15 T24 Odd-Osh- B) Veg T1 T2 T6 T9 T12 T15 T24 W Ddlta4 Dblp 99 Fig. 5. Southern blot hybridization analysis of Ddlta4 gene per haploid genome of D. discoideum. long of genomic DNA for D. discoideum was digested by BamH I (lane 1), EcoR I (lane 2), BamH I and EcoR I (lane 3), and Hind III (lane 4). The blots were probed with 3'ZP-radiolabeled 1.0 kb insert of the A ZAP Clone. Expected two bands were marked with arrows in EcoR I or Hind III cut lanes. 21Kb - 5.0Kb - 4.2kb - 100 1 2 3 - u)-- 101 transduction and are only present as intermediates. It is too early to say which possibility is real in D. discoideum. The Cloning of a full size CDNA and a sequence analysis remains to be done. To Check the LTA4 hydrolase activity, expression of Ddlta4 in E. coli can be done (Minami et al. 1988; Medina et 31., 1991). LTB4 is a potent chemoattractant for human blood polymorphonuclear leukocytes (Samuelsson et 31., 1987). It is not known whether LTB4 is a chemoattractant for D. discoideum. References Buttner, N ., Siegelbaum, S. A., and Volterra, A. Nature 342, 553-555 (1989). Crooke , S. T., Sarau, H., Saussy, D., Winkler, J., and Foley, J. Adv. Prostag. Throm. Leu. Res. 20, 127-137 (1990). Devereux J (1991) Program manual: Sequence Analysis Software Package, Version 7. Genetics Computer Group, Madison, Wisconsin Ford-Hutchison, A. W., Bray, M., Doig, M., Shipley, M., and Smith, M. Nature 286, 264-265 (1980) Funk, C. D., Radmark, O., Fu, J. Y., Matsumoto, T., Jomvall, H., Shimizu, T., and Samuelsson, B. Proc. Natl. Acad. Sci. USA 84, 6677-6681 (1987). Haeggsrom, J. Z., Wetterholm, A., Shapiro, R., Vallee, B. L., and Samuelsson, B. Biochim. Biophys. Res. Comm un. 172, 965-970 (1990). Hassanain, H. H., and Kopachik, W. J. Cell Sci. 94, 501-509 (1989). Irvine, R. F. Biochem. J. 204, 3-16 (1982). 102 Kim, D., Lewis, D. L., Graziadei, L, Neer, E. J ., Bar-Sagi, D., and Clapham, D. E. Nature 337, 557-560 (1989). Kurachi, Y., Ito, H., Sugirnoto, T., Shimizu, T., Miki, I., and U1, M. Nature 337, 555-557 (1989). Makita, N., Funk, C. D., Irnai, E., Hoover, R. L., and Bard, K. F. FEBS Lett. 299, 273-277 (1992). Malfroy, B., Kado-Fong, H., Gros, C., Giros, B., Schwartz, J. -C., and Hellmis, R. Biochim. Biophys. Res. Commun. 161, 236-241 (1989). Medina, J. F., Radmark, 0., Funk, C. D., and Haeggstrom, J. Z. Biochim. Biophys. Res. Commun. 176, 1516-1524 (1991). Medina, J. F., Wetterholm, A., Radmark, O., Shapiro, R., Haeggstrom, J. Z., Vallee, B. L., and Samuelsson, B. Proc. Natl. Acad. Sci. USA 88, 7620-7624 (1991). Minami, M., Minami, Y., Emori, Y., Kawasaki, H., Ohno, S., Suxuki, K., Ohishi, N., Shimizu, T., and Seyama, Y. FEBS Lett. 229, 279-282 (1988). Minami, M., Ohno, S., Kawasaki, H., Radmark, O., Samuelsson, B., Jomvall, H., Shimizu, T., Seyama, Y., and Suzuki, K. J. Biol. Chem. 262, 13873-13876 (1987). Parker, C. W. A. Rev. Immun. 5, 65-84 (1987). Piomelli, D., Volterra, A., Dale, N., Siegelbaum, S. A., Kandel, E. R., Schwartz, J. H., and Belardetti, F. Nature 328,3 8-43 (1987). Samuelsson, B., Dahlen, S.-E., Lindgren, J .-A., Rouzer, C. A. and Serhan, C. N. Science 237, 1171-1176 (1987). Samuelsson, B. and Funk, C. D. J. Biol. Chem. 263, 19469-19472 (1989). Van den Bosch, H. Biochem. biophys. Acta 604, 191-246 (1980). 103 Wetterholm, A., Medina, J. F., Radmark, O., Shapiro, R., Haeggstrom, J. Z., Vallee, B. L., and Samuelsson, B. Proc. Natl. Acad. Sci. USA 89, 9141-9145 (1992). 104 Discussion Efforts are underway to sequence the genomic DNA of humans and other prokaryotic and eukaryotic organisms (Watson, 1990; Stevens, 1992; Fleischmann et al., 1995). The genome projects are Changing the way research is done in biological sciences. Although much progress in gene identification has occurred, in the future a bigger goal of understanding the function of known genes awaits. Access to the whole genomic sequence will have tremendous influence on applied and academic research. More disease-related genes may be discovered and used for gene therapy. It is expected that more productive and pathogenic resistant crops may result. Fundamental knowledge on how gene expression is regulated and the developmental process is controlled will also be gained. Dictyosteli um is not only a model system for the study of development, it is useful in the search for, and analysis of eukaryotic genes. The genome size is about 40 Mb which is less than 2% that of humans. It is easier to Clone genes because fewer CDNA or genomic Clones need to be screened. The short size of introns (about 100 nucleotides) permits easier isolation of full size genomic DNA. For example, rat cystathionine B synthase (CBS) genomic DNA is about 25 Kb however, the CDNA is only 1.7 Kb due to lengthy introns (Swaroop et al., 1992). In contrast, Southern blot analysis of Dictyostelium CBS suggested that the size is less than 4 Kb (Fig. 3 in Ch. 4) whereas the CDNA was 1.6 Kb. The 80-95% AT rich 5' untranslated sequences on mRNA help to identify coding regions. Because AT rich sequences are likely to have a stop codon, TAA (the only one used in 105 Dictyostelium), an ATG following TAA in the 5' end is a likely candidate for the initiation codon. The haploid life cycle is an advantage in analyzing the function of Cloned genes by gene targeting through homologous recombination. The formation of diploids between normal haploid cells allows this system to be used for parasexual genetic study. When cultures of two strains of the same mating type are mixed they occasionally fuse to form heterozygous diploids. Since the diploids are stable for a number of generations, mutations can be determined to be dominant or recessive. However, the 1 X 10'5 rate of forming diploids is low (Loomis, 1980) and infrequent mitotic recombination results in low- resolution mapping. A Dictyostelium physical map with about 30 different markers was constructed by use of YAC vectors (Kuspa et 31. 1992). From several CDNA-mRNA and single-copy DNA-mRNA hybridization studies (Firtel, 1972; Blumberg and Lodish, 1980, 1981; Jacquet et al., 1981) it is known that about 4000 to 5000 different mRNAs are present in growing cells and about 2000 to 3000 developmental stage genes are expressed in post-aggregation cells. Loomis (1978) Claimed that only 150 to 300 genes among 2000 to 3000 developmental stage genes are needed for all of development from biochemical and genetic evidence. He suggested that most developmental genes may play minor and supportive roles during development without affecting morphogenesis under standard laboratory conditions. Diverse ways are used for Cloning genes. Screening Dictyostelium genes by homology using other eukaryotic DNAs or 106 guessmers as probes and differential screening for the vegetative or developmental stage genes are typical methods. Since Dictyostelium has biased codon usage by favoring A or T in the wobble position in synonomous codons (Spudich, 1989) the low number of redundancy, which increase possibility of picking target gene specifically, is possible for designing guessmers. The recently invented restriction enzyme mediated integration (REMI) method is valuable in especially the Cloning of genes with roles in development. For yeast, Schiestl and Petes (1991) found the efficiency of transformation was about 7- fold higher in a presence of the restriction enzyme than in its absence . Kuspa and Loomis (1992) showed that introducing a restriction enzyme used to linearize a transformation plasmid into Dictyosteli um along with the plasmid DNA increases more than 20- fold the frequency of integration into genomic restriction sites recognized by the specific enzyme. This integration of vector DNA causes abnormal development when DNA is integrated into genes which have significant roles in development. The vector DNA is used for Cloning targeted genes and Cloned genes are used for complementation of mutant phenotypes. By using this method the lagC gene which is required for muticellular development (Dynes et‘ al., 1994) was Cloned. Isolation of a cytokinesis mutant showed the possibility of Cloning of some genes expressed in vegetative cells by REMI method (Adachi et al., 1994). This method, however, does not allow the identification of two Classes of genes: those essential for cell viability or these functionally or physically redundant. Although these genes may have significant roles in development, they will not be recovered. Recently Chang et al. (in press) used modified REMI 107 method, so called promoter trap, to Clone genes whose disruption produces no obvious phenotype. They transformed laCZ coding gene using REMI and laCZ expressing transformants on development to select developmentally induced promoter. This method may allow us to Clone developmentally induced genes no better than differential screening does. It, however, does not allow Cloning of specific genes which have significant roles in development. Anyway, if Ioomis (1978) is true there are many developmentally regulated genes to be Cloned by methods other than Classic REM]. My approach was different than described above and led to isolation of genes expressed both in growth and development. Two novel genes (EHJ-l and IFK) and five genes (Dblp, Dlta4, DdCBS, DrpsZ4, and Drpl7a) which have known homologs in other organisms were found. Anchored PCR and DDRT-PCR methods were used to done these genes. Guessmers from known conserved amino acid sequences and A ZAP DNA specific oligomers were used for anchored PCR with A ZAP CDNA library as template. The use of DDRT-PCR for Cloning of induced or repressed genes in early development was also successful. When RNA from vegetative and 3 h developmental cells was compared, two or three differentially expressed bands were found with only one primer pair. More differentially expressed bands were identified between RNA from vegetative and slug cells. In one trial, 2 or 3 repressed and induced bands were identified amongst 50 detectable bands (data not shown). Kopachik et al. (1985) found that about 3% of genes were induced in differential screening using solution hybridization which is similar to what I can get from DDRT—PCR with RNA from vegetative and slug. Bands 108 repressed on development were isolated and Northern blot analysis was done to Check if the expression of Cloned genes are reduced. EHJ-I and Drpl7a CDNA sequences were Cloned using DDRT-PCR. The successful use of the DDRT-PCR method opens the possibility for quickly and efficiently Cloning many developmentally regulated genes. Some genome projects involve sequencing cDNAs because the sequence encoding proteins are obtained with less cost. Dictyostelium has about 70-75 % single copy sequence of which about 30 % is expressed in cytoplasmic mRNA (Firtel and Bonner, 1972; Blumberg and Lodish, 1981). Therefore about 21 % (0.7 X 0.3) of total genome is expressed in cytoplasmic mRNA and the cost for whole cDNAs sequencing will be five times less than genomic DNAs. Although methods for equalization of cDNAs in a library (Ko, 1990; Takahashi and Ko, 1994) are possible, some rare and important cDNAs can be missed while many housekeeping gene sequences will still be repetitively sequenced. The use of DDRT-PCR methods, however, may avoid these problems. The DDRT-PCR approach could be used to find many genes whose mRNAs are induced or reduced by CAMP or otherwise regulated. The constitutively expressed genes will be displayed as equal intensity bands. The developmentally regulated genes will be displayed as bands with reduced or induced intensity. About 50 distinct bands per primer pair were displayed, therefore differential displays with only 160 primer pairs (50 X 160) are required for 8000 different cDNAs. In practice, however, isolation of developmental stage genes requires more primer pairs because constitutive bands overlap differentially expressed bands. Usually 109 the 300-500 bp inserts can be obtained by DDRT-PCR and used for hybridization to a YAC library. Use of DDRT-PCR has several advantages than other methods for cloning of useful markers. 1) This method will normalize cDNAs in a CDNA library. Although there will be a difference in bands intensity, abundant mRNAs will be shown as a single band like rare mRNAs. Theoretically, different types of cDNAs are displayed as individual bands if primer sequences are not repeated in the same CDNA. Therefore use of this method can avoid the problem of repetitive Cloning of house keeping genes. 2) More developmentally regulated genes including those essential for cell viability and functionally or physically redundant ones missed by the REMI-based method can be Cloned. For example, the Ga7 or Ga8 null mutants which did not show abnormal phenotype could be Cloned by the DDRT-PCR method. 3) This method will be fast and economical. The construction of several CDNA libraries for different developmental stages is not necessary. To include all different types of cDNAs for genes induced by CAMP, DIF or ammonia, three normalized CDNA libraries are needed. For the purpose of constructing better physical maps using DDRT-PCR, all that is needed is first strand CDNA with mRNAs from different stages of cells. Loomis and Smith (1990) suggested that comparison of amino acid sequences is more reliable than untranslated nucleic acid sequences for evolutionary comparisons (Fig. 1). They Claim that Dictyostelium is more Closely related to mammals than is yeast in contrast to the analysis based on 18S rRNA (McCarroll et al., 1983). 110 McCarroll et al. (1983) : Based on comparison of 188 rRNA. Mammals Dictyostelium I Saccharomyces cerevisiae Loomis and Smith (1990) : Based on eight different protein sequences Saccharomyces cerevisiae DiC tyostelium Mammals Fig. I. Parsirnony of three different organisms. 111 The homologous sequences for DdCBS, Drpl7a, and DrpsZ4 are known in yeast and mammalian systems. Without exceptions the Dictyosteli um sequences have greater identical amino acids throughout their entire length to homologs of mammals than they do to homologs in yeast (54 % vs. 49 % for DdCBS; 55 % vs. 48 % for Drpl7a; 73 % vs. 67 % for Drps24). Therefore the comparison of deduced amino acid sequences Cloned in this thesis supports Loomis and Smith's Claim. Since it is thought that Dictyostelium and mammals diverged 1000-1200 million years ago, the sequence identity is remarkabe. The essential role of these proteins might act as a intense selection pressure to keep these sequences conserved. As I mentioned in the introduction CAMP receptor linked G- proteins have main roles in signal transduction in Dictyosteli um. Eight clones for different types of Gas and one for GB are identified. A constitutive GB is thought to interact with eight developmentally regulated Gas for the formation of heterotrimeric G protein complexes. Surprisingly, however, the GB knock-out mutant is viable although the developmental processes are completely blocked. One possible explanation is that G-protein related signal transduction has no role in growth. The other explanation is that other protein(s) may substitute for the role of GB in growth. A possible candidate for that role is the GB-like protein. In this thesis a Dictyos teli um homolog of the GB -like protein (Dblp) was Cloned. Dblp mRNA is constitutively expressed (Fig. 4 in Ch. 2). Dblp has a weak homology (26 % over middle 200 amino acids) to the the Dictyostelium G13 (Lilly et 31. 1993). However, Dblp 112 has seven WD-40 repeats, which are considered as domains for protein-protein interaction, as do other G13 proteins. The rat G B-like protein is a known receptor for activated C- kinase (RACKI) whose proposed role is mobilization of activated protein kinase C (PKC) to target region. The proposed role of this protein in plants is different. In tobacco, the homolog of this protein is induced by treatment of auxin and may participate in regulation of cell division. However, whether the G B-like protein can substitute for the role of GB in both systems is unknown. Ron et 31. (1994) proposed a model, in which a similar sequence between RACKI and a pseudo-RACKI sequence in PKC is a possible contact site in mobilization of activated PKC. I identified a similar sequence shared between Dblp and Dictyostelium myosin heavy Chain kinase (MHCK; Ravid and Spudich, 1992), the only Cloned protein kinase C in Dictyostelium. MHCK is translocated to myosin in response to CAMP. Phosphorylation of the myosin heavy Chain inhibits myosin thick filament formation and leads to mobilization of myosin to the posterior cortex of the polarized cell during chemotaxis (Yumura and Fukui, 1985; Ravid and Spudich, 1989). Dblp may have a role in the translocation of MHCK. MHCK has the amino acid sequence VMIWHL (311-316) which is similar to VMVWQL (46-5 1) in Dblp. The homologous sequence falls in the C2 region of MHCK as does the pseudo-RACK sequence of rat PKC. In inactive form of MHCK, VMIWHL may be covered by a specific region of MHCK. In active form of MHCK upon CAMP stimulation, the specific region of MHCK may be bound by VMVWQL in Dblp (Fig. 5 in Ch. 2). To test protein protein interaction the yeast two-hybrid 113 system (Fields and Song, 1989; Vojtek et 31., 1993) can be used with MHCK and Dblp CDNA Clones. Another way is to Check if in vitro translated proteins from the two cDNAs bind. When an antibody for Dblp is available, immunofluorescent detection (Yumura and Fukul, 1985) can be used to see colocalization of Dblp and MHCK in the posterior cortex of polarized cell during chemotaxis occurs. The proposed role of Dblp in development does not explain why Dblp mRNA is present during the vegetative stage. Although the Dblp protein level was not measured, for the purpose of discussion I assume that mRNA presence means active protein presence. Vegetative and early developmental cells are chemotactic to folic acid (Tillinghast and Newell, 1987). Although MHCK is not present in vegetative cells (Ravid and Spudich, 1992), but several related MHCKs have been purified from Dictyostelium (Cote and Bukiejko, 1987). Therefore Dblp may have a role in folic acid mediated chemotaxis in translocation of other MHCKs. Dblp may interact with G as and form functional heterotrimeric G proteins as GB does. This functional redundancy could allow GB null cells to grow normally. In the past research on mammalian cells, the GB y subunit was assumed to have no role in signal transduction. Some evidence, however, now exists that GB 7 is directly involved in regulation of effector molecules in signal transduction. It is possible GB 7 and Dblp have different effectors in Dictyostelium development. Dblp may control the localization of MHCK and GB y may have other unknown effector molecules, therefore GB null cells showed abnormal development in the presence of Dblp. 114 Although leukotriene receptors and signal transduction processes have been extensively Characterized in mammalian cells, they were unknown in lower organisms such as Dictyostelium before a CDNA was Cloned which encodes an enzyme, leukotriene A4 hydrolase (Dlta4). Dlta4 mRNA level is about four fold reduced in 2h developmental cells and increased to vegetative cell levels at the time CAMP relays start. The level was gradually reduced up to 20 folds after aggregation. This expression pattern is not common. In most cases, mRNAs are gradually reduced or induced during development. The down and up regulation pattern suggests that Dlta4 may have a significant role in leukotriene-related signal transduction during growth and early development. Mutant cells made null for CARI which is highly expressed in cells on early developmental stage, fail to bind or sense CAMP and arrest in early development. The CARI null cell phenotype implied that leukotriene may not substitute for the role of CAMP. in signal transduction. Another possible explanation is that the leukotriene system is controlled by CARI related signal transduction. Therefore lack of CARI causes improper regulation of Dlta4 and blocking of leukotriene related signal transduction. However, since leukotriene A4 hydrolase has not only hydrolase but also peptidase activity in the mammalian system, it is unclear leukotriene signal transduction exists in Dictyostelium. To test if Ddlta4 has roles in LTA4 hydrolase and peptidase activity, expression of the full size CDNA for Ddlta4 in E. coli can be attempted (Minami et 31., 1988). LTA4 hydrolase converts LTA4 into LTB4 and LTB4 is known as a potent chemoattractant for the human 115 blood polynuclear leukocytes (Samuelsson et 31., 1987). Perhaps Dictyostelium secretes and responds to LTB4. Because Dlta4 is a single copy gene gene targeting is possible. The targeted mutant should be affected at the aggregation stage if leukotriene-related signal transduction is necessary. Although five Clones (Dblp, Ddlta4, DdCBS, DrpsZ4, and Drpl7a) reported here had 40-70 % sequence identity to known sequence in entire deduced peptide sequences, whether they have similar roles needs to be tested. Complementation of yeast mutants, when they exist, with Dictyostelium cDNAs can be done. For example, complementation of a yeast cystathionine B synthase mutant (Cherest et 31.,1993) by DdCBS CDNA can be tried. A 1.3 Kb CDNA (IFK) had a conceptual 355 amino acids open reading frame which was homologous at 50 % identity over 61-186 amino acids region (data not shown) to eukaryotic initiation factor 2 a (eIF 2 a) kinase done by Roussou et 31. (1988) and Meurs et al., (1990). Northern blot analysis suggested that full size IFK CDNA should be 4 Kb, whereas the human eIF 2 a kinase mRNA size is 2.5 Kb and the yeast is 5 Kb. IFK and human eIF 2 a kinase mRNA levels increase upon starvation (Meurs et 31., I990). eIF2a kinases have 11 conserved domains in their catalytic region (Wek et al., 1989; Meurs et 31., 1990). The first five conserved domains were identified in IFK. Another features are homopolymer repeats of Gln, Asn, and Thr in the N-terminal region shared those within several other Dictyosteli um protein kinases (Mann and Firtel, 1991). However, the sequences, except conserved domains, did not have significant 116 homology to that of eukaryotic initiation factor 2a (eIF2 a) kinase. It will be important to Check whether other conserved domains are present in the full size CDNA and if IFK has eIF2a kinase activity using in vitro-translated product as Chen et al.(1991) did for the rabbit homolog. A yeast eIF2a kinase (GCNZ) mutant may be complemented by the full size IFK CDNA. To determine the role of Cloned genes (IFK, EHJ-I, Dblp, Dlta4, and DdCBS) in growth and development I tried to block gene expression using antisense RNA. However, the antisense experiments were not informative possibly because not enough antisense RNA was produced. For the production of antisense RNA pDNeoH vector was used. The pDNeoII vector uses an actin 6 promoter to control A the expression of insert. Actin 6 mRNA is present at a very low level in vegetative cells, increased in pre-aggregated cells and reduced in post-aggregative cells (Knecht et al., 1986). The antisense RNA production pattern was the same as that of actin 6 mRNA. Northern blot analysis, however showed that less antisense RNA was present than endogenous RNA in the transformant cells except for the IFK antisense producing cells. IFK antisense transformants had two times more antisense RNA in vegetative and 10 times more in 3 h developing, cells. Although more antisense, than endogenous RNA, was produced the reduction of [FK RNA was not detected. Lilly et 31. (1993) reported similar failure of antisense experiments. In attempts to block expression of Dictyostelium GB using antisense RNA, endogenous GB mRNA was not reduced although more antisense RNA was produced than endogenous RNA. The reason for 117 failure is unknown, but inappropriate localization of endogenous RNA is a possibility. Scherczinger et 31. (1992) analyzed variables affecting antisense RNA inhibition of gene expression. They showed that the critical factor for the inhibition of myosin heavy Chain II (MHC II) gene expression by antisense RNA was the particular fragment of the gene used to produce the antisense. The fragments that produce the greatest inhibition were from the 3' end region of the gene. About 400 bp Chosen at random amongst 8000 bp did not inhibit the expression of MHC II gene expression. If Scherczinger et 31.'s Claim (1992) is true, the failure of antisense experiment in my case can be explainable. I used 5' end of cDNAs when 1 construct antisense RNA producing vector and the size of DNA was about 500 bp in every case. Although many successful antisense RNAs are from other than 3' end or short oligomers in other systems (Crowley et al. 1985; Watkins et 31., 1992), 3' end or full size cDNAs or genomic DNA can be used again for antisense experiments in my case. An advantage of working with Dictyostelium is having a relatively simple and easy method for isolation of gene targeted cells. Genes for CAMP receptors, Ga, GB and protein kinase were targeted and their roles studied. Gene targeting of my Cloned genes will give significant information for the function of these genes. The knock- out cells of Dblp or Dlta4 may arrest at early developmental stages. The function of EHJ-l is obscure. I will get some idea for the role of EHJ-I in growth and development if null cells have detectable phenotypic Changes. Although genes expressed in growth are not all 118 essential for cell viability, IFK, Dblp, Dlta4, DdCBS and EHJ-I knock- out cells will not be found if they are. To circumvent this problem, the pVEII vector, which uses the discoidin I gamma promoter for expression of genes (Blusch et al., 1992), or the use of the Cre/loxP recombination system, allowing inducible deletion in mice (Gu et al., 1994; Kuhn et al., 1995), are alternatives. Inducible expression will be useful to examine the effects of protein overexpression or antisense-mediated down-regulation since it allows for a direct comparison of the 'on' and 'off‘ state of the promoter. The discoidin I gamma promoter is inducible by folate and the expression of antisense RNA can be controlled by folate. The growth rate and development would be Checked in antisense producing cells. To avoid the problem of not enough expression of antisense RNA, 3 discoidin overproducer mutant can be used (Wetterauer et 31. 1993). The discoidin promoter is overexpressed by a factor of 10 to 100 under control by folate as in wild type cells. Thus the pVEII vector allows overproduction of inducible antisense RNA or proteins. A recombinase called Cre from bacteriophage P1 deletes any phage genome integrated into Chromosomes. Cre lines up short sequences of phage DNA called loxP sites and removes the DNA between them, leaving one loxP site behind. The Cre/loxP recombination system works in transgenic mice. Gu et al. (1994) and Kuhn et 31. (1995) demonstrated that a target gene flanked with loxP sequences could be eliminated by conditionally induced Cre. If the Cre/loxP recombination system works in Dictyostelium, inducible gene targeting may be possible. The expression of Cre gene could be 119 controlled by the discoidin promoter and target genes flanked by loxP sequences in the Cre+ cells. Removal of folic acid from growth media or initiation of development will cause deletion of the target gene and the role of that gene in growth or development can be studied. The overexpression of genes causes phenotypic Changes in several cases (Simon et al., 1989; Faix et al., 1990). For example, the overexpression of regulatory (R) subunit of CAMP dependent protein kinase in vegetative cells when endogenous R subunit is not normally present causes an inability of cells to aggregate (Simon et 31., 1989). In my research, constitutive overexpression of EHJ-I leads to delay of deve10pment and formation of smaller fruiting bodies. It is not Clear how overexpression of EHJ-l causes that phenotypic Changes. Several possible explanations for this phenotype Change were described in Chapter one. In summary, I cloned and sequenced several cDNAs with new approaches and Characterized the regulation of mRNA expression for those clones. Conserved sequences in signal transduction-related genes of other systems were identified in Dblp and Dlta4 and the role of those genes were described. The comparison of deduced peptide sequences implied that Dictyostelium is more Closely related to mammals than is yeast. The next step should be determination of the role of these Cloned genes. This research opens several possibilities which were not recognized so far in Dictyosteli um research. 120 References Adachi, H., Hasebe, T., Yoshinaga, K., Ohta, T., and Sutoh, K. (1994). Biochim. Biophys. Res. 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Nature 314, 194-196. 123 APPENDIX I : Primary sequence and developmental regulation of Dictyostelium discoideum ribosomal protein 824 and L7a mRNA The work for Drp824 in appendix I was submitted to gene. 124 Introduction In eukaryotes about 70 different types of ribosomal proteins are part of the functional ribosome (Warner, 1989). The expression of ribosomal proteins and formation of ribosomes are controlled according to the rate of cell growth and developmental stages (Angerer et al., 1992; Ken and Singleton, 1994). Dictyostelium discoideum, a cellular slime mold, initiates development upon starvation. During development expression of several mRNAs for D. discoideum ribosomal protein is reduced (Steel and Jacobson, 1988; Singleton et al., 1989; Proffitt et al., 1991; for review, Ramagopal, 1992). In D. discoideum development CAMP acts as a signaling molecule and regulates many developmentally expressed genes (Kimmel, 1987; Mann and Firtel, 1987). Two D. discoideum cDNAs which appear to encode a protein homologous to the yeast 40S ribosome protein 524 and the human 605 ribosomal protein L7a were cloned and sequenced. Results and Discussion 1) Homolog to the yeast 40S ribosomal protein S24 The CDNA would encode a protein of 130 amino acids with a molecular weight of 14,895 daltons (Fig. 1). The protein has a net positive Charge of +10.3 as do other 524 ribosomal proteins (Bonham- Smith et al., 1992; Angerer et al., 1992). The protein may correspond to 517 or S18 of the nomenclature for Dictyostelium ribosomal protein when the size and p1 are considered (Ramagopal and Ennis, 125 Fig. 1. Nucleotide sequence of D. discoideum ribosomal protein DrpsZ4. The sequence was determined by the dideoxy techniques using Sequenase 2.0 (United States Biochemical). The putative polyadenylation site was underlined. 126 10 3O 50 GCACGAGG'I'I‘A'I‘ACT'I‘GAAACAGATACAAAA‘IGGTCAGAA'I‘CAG'IG'I‘T'I'I‘AAACGA‘I'I‘GC MetValArgIleSerValLeuAsnAsprs 70 90 110 TTATACTCCA‘I‘TGTCAA'I‘GC CGAAAGACAAGGTAAAAGACAAGTCT‘I‘AGTCAGACCATCA LeuTyrSerIleValAsnAlaGluArgGlnGlyLysArgGInValLeuValArgProSer 130 150 170 TCAAAAGTCATCGTTAAATTCTTAGAAG'I'I‘ATGA'I‘GAAAAAGAGATACA‘I'I'GGTGAA’ITC SerLysValIleValLysPheLeuGluValMetMetLysLysArgTyrIleGlyGluPhe 190 210 230 GAAATCGTTGATGACCATCGTTCCGGTAAAATTGTCATTGATTTAATCGGTCGTATCAAC GluIleValAspAspHisArgSerGlyLysIleValIleAspLeuIleGlyArgIleAsn 250 270 290 AAATGTGGTGTCATCTCCCCAAGATTTGACGTTACTTTAGACGAAATCGAAAAATGGGCC LysCysGlyValI1eSerProArgPheAspValThrLeuAspGluIleGluLysTrpAla 310 330 350 TCTTACTTACTCCCATCCCGTCAATTCGGTCATATCGTCCTCACCACCTCCCTCGGTATC SerTereuLeuProSerArgGlnPheGlyHisI1eValLeuThrThrSerLeuGlyI1e 370 390 410 ATGGACCACAACGAAGCCAAAACCAGACACACTGGTGGTAAATTATTAGGTTTCTTCTAT MetAspHisAsnGluAlaLysThrArgHisThrGlyGlyLysLeuLeuGlyPhePheTyr 430 450 470 TAAATTGCTAGTCTPITITWTPAAAATATTATPTAAATCCTPTAAAAAAAAAA AAAAAAAA 127 1980). From the homology to the yeast ribosomal protein subunit S24, the CDNA was named Dictyostelium ribosomal protein SL4 (DrpsZ4). The consensus polyadenylation signal AATAAA was found at 442-447 nucleotides (Steel et 31., 1987). The deduced peptide sequence (Fig. 2) showed 72% sequence identity (85% similarity) to sea urchin (Angerer et al., 1992), and 67% identity (82% similarity) to yeast, ribosomal protein (Leer et al., 1985). An autoradiograph of a northern blot shows a 0.5 kb band which is similar to the size of Drps24 CDNA. Drp524 mRNA level was highest in actively growing cells and reduced up to 10 fold in cells developing an agar plate (Fig. 3A). To Check if Drps24 mRNA level was regulated by extracellular CAMP during development, we isolated total RNA from cells shaking in DB buffer with or without a CAMP pulse as described (Hassanain and Kopachik, 1989). The Drps24 mRNA level was reduced in cells given CAMP (Fig. 3B). The GenBank/EMBL accession number for D. discoideum Drps24 is U27539. 2) Homolog to the mouse 60S ribosomal protein L7a The A ZAP Clone which has about 1.0 Kb DNA insert was Cloned and sequenced (Fig.4). Its conceptual translation product contains an open reading frame with 278 amino acids. The deduced peptide sequence (Fig.5) has 55% identity (68% similarity) to the human (Ziemiecki et al., 1988), and 48% identity (64% similarity) to yeast, ribosomal protein L7a (Arevalo and Warner, 1990). Due to the significant homology to L7a this CDNA was named Dictyostelium ribosomal protein 113 (Drpl7a). Northern blot data suggest that the 128 Fig. 2. Alignment of RPSZ4 sequences. DrpsZ4, D. discoideum ribosomal protein 824 (this report); YS24, yeast ribosomal protein YS24 (Leer et al., 1985); rpslSa, Brassica napus ribosomal protein rpslSa (Bonham-Smith et 31., I992); SpS24, Sea urchin ribosomal protein SpSZ4. The alignment was optimized by the Pileup program (The University of Wisconsin Genetics Computer Group software, Devereux, 1991). 0, residues strictly conserved in the four sequences. Drp524 YSZ4 rpslSa Sp324 Drp524 YSZ4 rpslSa SpSZ4 Drp524 YSZ4 rpslSa Sp824 1 MVRISVLNDC MTRSSVLADA MVRISVLNDA MVRMNVLADA 51 EIVDDHRSGK EYIDDHRSGK EYVDDHRSGK EIVDDHRGGK 101 HIVLTTSLGI YVILTTSAGI YIVLTTSAGI YVVLTTSGGI 12$) LYSIVNAERQ LNAINNAEKT LKSMFNAEKR LRSICNAEKR IVIDLIGRIN IVVQLNGRLN IVVELNGRLN IIVNLNGRLN MDHNEAKTRH MDHEEARRKH MDHEEARRKN MDHEEARRKH GKRQVLVRPS GKRQVLIRPS GKRQVMIRPS CKRQVLIRPC KCGVISPRFD KCGVISPRFN KCGVISPRFD KCGVISPRFD 130 TGGKLLGFFY VSGKILGFVY VGGKVLGFFY VGGKILGFFF SKVIVKFLEV SKVIIKFLQV SKVIIKFLIV SKVTVKFLMV VTLDEIEKWA VKIGDIEKWT VGVKEIEGWT VPINEMEKWT 50 MMKKRYIGEF MQKHGYIGEF MQKHGYIGEF MMKHGYIGEF 100 SYLLPSRQFG ANLLPARQFG ARLLPSRQFG SNLLPSRQFG 130 Fig. 3. Expression of Dictyostelium Drps24 transcripts. A) RNA was isolated from vegetative cells and during development on agar for 3, 6, 9, 12, 15 and 24h. pLK326 mRNA was used to show equivalent amount of intact mRNA were loaded in each lane. B) Vegetative amoebae were resuspended at 2x107 cells /ml in DB buffer and shaken at 250rpm for 10 h without CAMP addition (T10S) or for four h at which time 50 nM CAMP was given every 10 min for 6 h (T10P). The blots were probed with 32F radiolabeled insert of the A ZAP Clone. 131 A) Veg T3 T6 T9 T12 T15 T24 ...H.' '0 C-F-Dw B) TlOS TIOP Q... "U Drp824 pLK326 132 Fig. 4. Nucleotide and deduced peptide sequence of D. discoideum ribosomal protein Drpl7a. The putative polyadenylation site was underlined. 133 10 3O 50 CACGAGGCTGCCACCAAAGCTGCTCCAGCCAAAACCGCTGTTGCCACCACCAAATCAAAG HisGluAlaAlaThrLysAlaAlaProAlaLysThrAlaValAlaThrThrLysSerLys 7O 90 110 AAAGTCGTCAAGAAGGGTGAGAAGAAAATCAAAACTAGAACCTTATTCAACGCCTTATAC LysValValLysLysGlyGluLysLysIleLysThrArgThrLeuPheAsnAlaLeuTyr 130 150 170 ACCAAAAACGTCAAAAACTTTGGTACTGGTTTCGGTGTTCAACCAAAGAGAGATTTAACT ThrLysAanalLysAsnPheGlyThrGlyPheGlyValGlnProLysArgAspLeuThr 190 210 230 CATTTCACTCACTGGCCAAGATACATCAAATTACAAAGACAAAGACGTGTTTTATTAAAG HisPheThrHisTerroArgTyrIleLysLeuGlnArgGlnArgArgValLeuLeuLys 250 270 290 AGATTAAAGGTTCCACCAACAATCAACCAATTCACCCGTGTCTTTGACAAAAACACCGCT ArgLeuLysValProProThrIleAsnGlnPheThrArgValPheAspLysAsnThrAla 310 330 350 GTCCATTTATTCAAATTATTAGATAAATACAGACCAGAAGAAGCCTCAGTCAAGAAAGCT Va1HisLeuPheLysLeuLeuAspLysTyrArgProGluGluAlaSerValLysLysAla 370 390 410 AGATTATTGAAAATCGCTGAAGCCCGTGCTGCCACTCCAAAAGGTCAAGCTGCTCCAAAA ArgLeuLeuLysIleAlaGluAlaArgAlaAlaThrProLysGlyGlnAlaAlaProLys 430 450 470 GCTGAAAAACCAGTCCGACACTTACGTTTCGGTATTAACTCTGTCACCAAATTAATCGAA AlaGluLysProValArgHisLeuArgPheGlyIleAsnSerValThrLysLeuIleGlu 490 510 530 AAGAAGAAAGCTAAATTAGTCGTCATTGCCCACGATGTTGACCCAGTTGAACTCGTCTTA LysLysLysAlaLysLeuValValIleAlaHisAspValAspProValGluLeuValLeu 550 570 590 TACATACCAACCCTCTGCAGACGTATGGATGTCCCATACTGTATCGTCAAATCTAAATCC TyrIleProThrLeuCysArgArgMetAspValProTerysIleValLysSerLysSer 610 630 650 AGATTAGGTGAATTAGTTCACATGAGAAACGCTTCATGTGTTGCCCTCACTGGTGTCAAC ArgLeuGlyGluLeuValHisMetArgAsnAlaSerCysValAlaLeuThrGlyValAsn 670 690 710 TCTGCTGACTCAAACGAACTCGCTTTATTAGTTGAATCCGCCAAACAAATGTTCGACAAT SerAlaAspSerAsnGluLeuAlaLeuLeuValGluSerAlaLysGlnMetPheAspAsn 730 750 770 AACAGTGAACACAGAAAGACCTGGGGTGGTAACACTTTATCTGGTCCAGCTCGTGCTATC AsnSerGluHisArgLysThrTrpGlyGlyAsnThrLeuSerGlyProAlaArgAlaI1e 790 810 830 TTAGCCAAACGTCAAAAAGCTGAAGCCAAAGAAAGTTTAGCCAAATCAAAGATCTAAGCT LeuAlaLysArgGlnLysAlaGluAlaLysGluSerLeuAlaLysSerLysIle 850 870 890 CTTTAATAGTTTAGCGAGACTCTCACTCTTTTTTTAAATAATCAAATAAAATAAAGGTTC 910 930 950 TTTAAAAAAAAAAAATACTAAAAAAAAAATTTTATTCTTTTAAAAAAAAAAATAAAAAAA 970 AAAAAAAAAAAAAA 134 Fig. 5. Alignment of RPL7a sequences. L7aDd, D. discoideum ribosomal protein L7a (this report); L7aChiCken, Chicken ribosomal protein L7a (Maeda et al., 1993); L7aHuman, the human ribosomal protein L7a (Ziemiecki et al., 1988); L7aRice, rice ribosomal protein L7a (Nishi et 31., 1993); L7aYeast, yeast ribosomal protein RPL4A (Arevalo and Warner, 1990). The alignment was optimized by the Pileup program (The University of Wisconsin Genetics Computer Group software, Devereux, 1991). 0, conserved residues in all sequences or in four sequences including L7aDd. L7aDd L7aChicken L7aHuman L7aRice L7aYeast L7aDd L7aChicken L7aHuman L7aRice L7aYeast L7aDd L7aChicken L7aHuman L7aRice L7aYeast L7aDd L7aChicken L7aHuman L7aRice L7aYeast L7aDd L7aChicken L7aHuman L7aRice L7aYeast L7aDd L7aChicken L7aHuman L7aRice L7aYeast ....HEAATK PKGKKAKGKK PKGKKAKGKK ...... MAPK 51 FGTGFGVQPK FGIGQDIQPK FGIGQDIQPK FGIGGALPPK FGIGQAVQPK 101 KNTAVHLFKL RQTATQLLKL RQTATQLLKL KNLATNLFKM RNTAAETFKL 151 HLRFGINSVT .LRAGVNTVT .LRAGVNTVT .VKYGLNHVT .VKYGLNHVV 201 KSKSRLGELV KSKARLGRLV KGKARLGRLV KGKARLGSIV KGKARLGTLV 251 KTWGGNTLSG RHWGGNVLGP RHWGGNVLGP KKWGGGVMGS KHWGGGILGN lj35 0.. AAPAKTAVAT VAPAPAVVKK VAPAPAVVKK RGGRAPVPAK VAPAPFGAKS RDLTHFTHWP RDLTRFVKWP RDLTRFVKWP KDLHRFVKWP RNLSRYVKWP LDKYRPEEAS AHKYRPETKQ AHKYRPETKQ LLKYRPEDKA FNKYRPETAA KLIEKKKAKL TLVENKKAQL TLVENKKAQL YLIEQSKAQL SLIENKKAKL HMRNASCVAL HRKTCTCVAF HRKTCTTVAF HKKTASVLCL NQKTSAVAAL PARAILAKRQ KSVARIAKLE KSVARIAKLE KSQAKTKARE KAQAKMDKRA TKSKKVVKKG QEAKKVV... QEAKKVV... KKTEKVT... TKSNKAK... RYIKLQRQRR RYIRLQRQRS RYIRLQRQRA KVVRIQRQRR EYVRLQRQKK VKKARLLKIA EKKQRLLARA EKKQRLLARA AKKERLLKRA EKKERLTKEA VVIAHDVDPV VVIAHDVDPI VVIAHDVDPI VVIAHDVDPI VLIANDVDPI TGVNSADSNE TQVNPEDKGA TQVNSEDKGA TTVKNEDKLE TEVRAEDEAA KAEAKESLAK KAKAKELATK KAKAKELATK KLLAKEAAQR KTSDSA.... EKKIKTRTLF .......... cccccccccc oooooooooo oooooooooo VLLKRLKVPP ILYKRLKVPP ILYKRLKVPP ILKQRLKVPP ILSIRLKVPP O O EARAATPKGQ EQKAAG.KGD EKKAAG.KGD QAEAEG.KT. AAIAEG.KSK ELVLYIPTLC ELVVFLPALC ELVVFLPALC ELVVWLPALC ELVVFLPALC LALLVESAKQ LAKLVEAVKT LAKLVEAIRT FSKILEAIKA LAKLVSTIDA 283 SKI LG. LG. 50 NALYTKNVKN NPLFEKRPKN NPLFEKRPKN NPLFEKRPKQ NPLTHSTPKN 100 TINQFTRVFD AINQFSQALD AINQFTQALD ALNQFTRTLD TIAQFQYTLD 150 O O AAPKAEKPVR TPTK.RPPV. VPTK.RPPV. VEAK.KPIV. QDASPKPYA. 200 RRMDVPYCIV RKMGVPYCII RKMGVPYCII RKMEVPYCIV KKMGVPYAII 250 MF.DNNSEHR NYNDRYDEIR NYNDRYDEIR NFNDKFDEVR NFADKYDEVK 136 full size CDNA should be about 1.2 Kb. The 1.0 Kb insert DNA appears to lack of an initiation codon. The consensus polyadenylation signal AATAAA was found at 885-890 nucleotides (Steel et al., 1987). Northern blot analysis showed that the same pattern of mRNA expression as Drps24 (data not shown). Acknowledgment A A zap CDNA library made to vegetative cell mRNA was a generous gift from Dr. Herbert L. Ennis ( Roche Institute of Molecular Biology ). References Angerer,L.M., Yang,Q,, IiesveldJ., Kingsley,P.D., and Angerer,.R.C.: Tissue-restricted accumulation of a ribosomal protein mRNA is not coordinated with rRNA transcription and proceeds growth of sea urchin pluteus larva. Dev. Biol. 149 (1992) 27-40. Arevalo, S. G., and Warner, J. R.: Ribosomal protein L4 of Saccharomyces cerevisiae: the gene and its protein. Nucleic Acids Res. 18 (1990) 1447-1449. Bonham-Smith,P.C., Oancia,T.L., and Moloney,M.M.: Cytoplasmic ribosomal protein 5153 from Brassica napus:: Molecular Cloning and developmental expression in mitotically active tissues. Plant Mol. Biol. 18 (1992) 909-919. Devereux J (1991) Program manual: Sequence Analysis Software Package, Version 7. Genetics Computer Group, Madison, Wisconsin 137 Hassanain, H. H., and Kopachik, W.: Regulatory signals affecting a selective loss of mRNA in Dictyosteli um discoideum. J. Cell Sci. 94 (1989) 501-509. leer, R. J ., van Raamsdonk-Duin, M.M.C., Kraakman, P., Mager, W.H., and Planta, R.J.: The genes for yeast ribosomal proteins 824 and L46 are adjacent and divergently transcribed. Nucleic Acids Res. 13 (1985) 701-709. Ken, R. and Singleton, C. K.: Redundant regulatory elements account for the developmental control of a ribosomal protein gene of Dictyostelium discoideum. Differentiation 55 (1994) 97-103. Kimmel, A. R.: Different molecular mechanisms for CAMP regulation of gene expression during Dictyosteli um development. Dev. Biol. 122 (1987) 163-171. Maeda, N ., Kenmochi, N., and Tanaka, T.: The complete nucleotide sequence of Chicken ribosomal protein L7a gene and the multiple factor binding sites in its 5'—flanking region. Biochimie. 75 (1993) 785-7 90. Mann, S. and Firtel, R. A.: Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor. Mol. Cell. Biol. 7 (1987) 45 8-469. Nishi, R., Kidou, S., Uchimiya, H., and Kata, A. : The primary structure of two proteins from the large ribosomal subunit of rice. Biochim. Biophys. Acta. 1216 (1993) 110-112. Proffitt, J. A., Jagger, P. S., Wilson, G. A., Donovan, J. T., Widdowson, D. C. and Hames, B. D.: A developmentally regulated gene encodes the Dictyosteli um homolog of yeast ribosomal protein S4 and 138 mammalian LLRep3 proteins. Nucleic Acids Res. 19 (1991) 3867- 3873. Ramagopal, S.: The Dictyostelium ribosome: biochemistry, molecular biology, and developmental regulation. Biochem. Cell Biol. 70 (1992) 738-750. Ramagopal, S. and Ennis, H. L.: Studies on ribosomal proteins in the cellular slime mold Dictyosteli um discoideum. Resolution, nomenclature and molecular weights of proteins in the 40S and 605 ribosomal subunits. Eur. J. Biochem. 105 (1980) 245-258. Singleton, C. K., Mannings, S. S. and Ken, R.: Primary structure and regulation of vegetative specific genes of Dictyostelium discoideum. Nucleic Acids Res. 17 (1989) 9679-9692. Steel, L. F. and Jacobson, A.: Post-transcriptional regulation of ribosomal protein gene expression during deve10pment in Dictyostelium discoideum. Dev. Gen. 9 (1988) 421-434. Steel, L. F., Smyth, A., and Jacobson, A.: Nucleotide sequence and Characterization of the transcript of Dictyostelium ribosomal protein gene. Nucleic Acids Res. 15 (1987) 10285-10298. Warner, J .R.: Synthesis of ribosomes in Saccharomyces cerevisiae. Microbiol. Rev. 53 (1989) 256-271 Ziemiecki, A., Mueller, R. G., Hynes, N. E., Krieg, J ., and Kozma, S. C. : CDNA sequence of the human ribosomal large subunit protein L7a. Nucleic Acids Res. 16 (1988) 10356 139 Appendix II : Cloning and Characterization of Dictyostelium discoideum CDNA encoding cystathionine B-synthase Chapter four was submitted to Gene. 140 INTRODUCTION Cystathionine B-synthase (CBS, EC 4.2.1.22) is a key enzyme for cysteine and methionine biosynthesis in eukaryotic systems (Griffith, 1987). In a pyridoxal 5'-phosphate (PLP) dependent manner CBS catalyzes the condensation of homocysteine with serine to form cystathionine. Cystathionine is converted to cysteine by another PLP dependent enzyme cystathionine y-lyase (EC 4.4.1.1). CBS deficiency in humans is an autosomal recessive disease, homocystinuria (Mudd et 31,1989). Dictyostelium discoideum is a soil amoeba which feeds on bacteria. When the food source is depleted, the cells initiate a program of multicellular development (Loomis, 1982). Four h after removal of the food source, a small percentage of starving cells begin emitting pulses of cyclic adenosine monophosphate (CAMP). Neighboring cells sense CAMP via CAMP receptors expressed on the cell surface, move chemotactically towards the source and relay the CAMP signal. By nine h, a tight aggregate of cells is established. Differentiation continues with formation of a tipped aggregate at about 12 h, a migrating slug at about 16 h, and a mature fruiting body at 24h (Firtel, 1991). Here we report the D. discoideum sequence of a CDNA encoding cystathionine B-synthase and the regulation of the mRNA during development. EXPERIMENTAL AND DISCUSSION 141 (a) Cloning and sequence analysis Among a group of developmentally regulated genes we isolated a partial CDNA whose deduced amino acid sequence showed strong homology to human and rat cystathionine B-synthase. By using the CDNA insert as a probe of a CDNA library made to vegetative cell mRNA we isolated a plasmid with a CDNA 1.6kb insert. A contiguous 1,611 bp stretch of DNA was sequenced (Fig.1). The nucleotide sequence contains a single open reading frame (ORF) of 1,491 bp. Conceptual translation of the ORF gives a protein of 497 amino acid residues, with a calculated Mr of 54,400. Potential AATAAA polyadenylation signals were found (Steel et al., 1987) A GenBank database search with the FastA program showed that its deduced amino acid sequence had 54% identity and 71% similarity over its entire length with the human CBS (Fig.2). Yeast CBS showed 49% identity and 65% similarity to DdCBS(Fig.2). DdCBS also had significant homology (37 and 39% identity) with bacterial cysteine synthase A and B encoded by cysK and cysM (Byrne et al., 1988; Sirko et 31.,1990). DdCBS has a putative pyridoxal phosphate attachment site (Lysine 73) which is used as a cofactor for CBS and cysteine synthase. (b) Southern blot analysis To Check the copy number of the DdCBS gene, Southern hybridization analysis was performed. Genomic DNA was isolated from D. discoideum strain KAX4 and digested with several different enzymes. Labeled probes from the 1.6 kb insert or EcoRI/Sall fragment (1—1088bp) were found to hybridize to a single restriction 142 Fig. 1. Nucleotide sequence of the D. discoideum DdCBS gene and deduced amino acid sequence. Putative PLP binding sites are marked by bold letters at amino acids 62 and 73. Potential overlapped polyadenylation sites were underlined from base 15 27 to 1542. The sequence of the DdCBS CDNA was determined by the dideoxy-termination method using synthetic oligonucleotide primers and the Sequenase DNA sequencing kit (United States Biochemical). 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1421 1481 1541 143 ACGAGTTTTTTTTTTTTTTTAAACCTCTCTAAAACAAACAAATAACTAAAAATGTCAGCA M S A CCAGAAGGACCATCAAAATGCACTTGGACTCCAAATACCACTGAAAACACTCCACATACC P E G P S K C T W T P N T T E N T P H T ACCAGAAGAACTCCAAAGAAATTAATTATGGATAATATTCTTGATAATATTGGTGGAACA T R R T P K K L I M D N I L D N I G G T CCATTAGTTAGAGTTAATAAAGTTTCATCAGATTTAGAATGTGAATTAGTTGCAAAATGT P L V R V N K V S S D L E C E L V A K C GAATTTTTCAATGCAGGTGGTTCAGTTAAGGATCGTATTGGTCATCGTATGATTGTTGAT E F F N A G G S V K D R I G H R M I V D GCAGAAGAGAGTGGTAGAATTAAGAAAGGAGATACATTAATTGAACCAACCTCTGGTAAC A E E S G R I K K G D T L I E P T S G N ACTGGTATTGGTTTAGCATTGACAGCAGCCATCAAAGGTTACAAAATGATCATTACACTC T G I G L A L T A A I K G Y K M I I T L CCAGAGAAAATGTCACAAGAGAAAGTTGATGTCTTGAAAGCATTGGGAGGAGAGATCATT P E K M S Q E K V D V L K A L G G E I I CGTACACCAACTGAAGCAGCATTTGATGCACCAGAGTCACATATTGGTGTTGCAAAGAAA R T P T E A A F D A P E S H I G V A K K TTAAATTCAGAGATTCCAAATTCCCACATTTTAGATCAATACGGTAACCCATCCAATCCA L N S E I P N S H I L D Q Y G N P S N P TTGGCCCATTACGATGGTACCGCCGAAGAACTCCTCGAACAATGTGAGGGTAAGATTGAT L A H Y D G T A E E L L E Q C E G K I D ATGATCGTTTGCACAGCCGGTACCGGTGGTACAATCACTGGTATTGCCAGAAAGATCAAA M I V C T A G T G G T I T G I A R K I K GAAAGACTTCCAAACTGTATCGTCGTTGGTGTCGATCCACATGGTTCAATTCTCGCTCAA E R L P N C I V V G V D P H G S I L A Q CCAGAATCACTCAACAATACCAACAAGAGTTACAAAATCGAAGGTATCGGTTACGATTTC P E S L N N T N K S Y K I E G I G Y D F ATTCCAAACGTTCTCGAACGTAAATTAGTCGATCAATGGATCAAAACCGACGATAAGGAA I P N V L E R K L V D Q W I K T D D K E TCTTTCATCATGGCTCGTCGTCTCATTAAAGAAGAAGGTCTCCTTTGCGCTGGTAGTTCA S F I M A R R L I K E E G L L C A G S S GGTTCCGCTATGGTTGGTGCACTCCTAGCCGCCAAACAATTGAAAAAAGGTCAACGTTGT G S A M V G A L L A A K Q L K K G Q R C GTTGTCTTATTAGCCGATTCCATTAGAAACTATATGACCAAACATTTAAATGATGATTGG V V L L A D S I R N Y M T K H L N D D W TTAGTCGACAATGGTTTCGTTGATCCAGAATACAAAACTAAAGATCAACAAGAAGAAGAG L V D N G F V D P E Y K T K D Q Q E E E AAATATCATGGTGCCACCGTCAAAGATTTAACACTCCCAAAACCAATCACCATCTCTGCC K Y H G A T V K D L T L P K P I T I S A ACCACCACTTGTGCTGCCGCAGTTCAACTCCTCCAACAATATGGTTTCGATCAATTACCA T T T C A A A V Q L L Q Q Y G F D Q L P GTCGTTAGTGAATCAAAAAAAGTTTTGGTCAACTCACTCTTGGTAACTTCTCTCACATAT V V S E S K K V L V N S L L V T S L T Y GCCTCTAAAAAAGCTGTCCCAACTGATGCTGTCAGTAAAGTTATGTTCCGTTTCACTAAA A S K K A V P T D A V S K V M F R F T K AATGAAAAATATATTCCAATCACTCAATCAACTTCTTTAGCTACTCTTAGCAAATTTTTC N E K Y I P I T Q S T S L A T L S K F F GAAAATCATAGCAGTGCTATCGTAACTGAAAATGATGAAATCATTTCAATTGTAACTAAA E N H S S A I V T E N D E I I S I V T K ATTGATTTATTAACTTATTTAATGAAATCTCAACAAAAAAATTAAAAATAAAATAAAATA I D L L T Y L M K S Q Q K N AAAAAAAAAACACGTAATATTAATAATAATTATAAAAAAAAAAAAAAAAAA 23 43 63 83 103 123 143 163 183 203 223 243 263 283 303 323 343 363 383 403 423 443 463 483 497 144 Fig. 2. Alignment of cystathionine B-synthase sequences. CBS-DIC, D. discoideum cystathione B-synthase (this report); CBS- HUMAN, human cystathionine B-synthase (Kruger and Cox, 1994); CBS-RAT, rat cystathionine B-synthase (Swaroop et al., 1992; Ishihara et al., 1990); CBS-YEAST, cystathionine encoded by the S.cerevisi3e STR4 gene (Cherest et al., 1993). The alignment was optimized by the Pileup program (The University of Wisconsin Genetics Computer Group software, Devereux et al., 1984): 0) residues strictly conserved in the four sequences 0) residues conserved in three sequences only #) residues similar in four sequences. CBS-DIC CBS-HUMAN CBS-RAT CBS-YEAST CBS-DIC CBS-HUMAN CBS-RAT CBS-YEAST CBS-DIC CBS-HUMAN CBS-RAT CBS-YEAST CBS-DIC CBS-HUMAN CBS-RAT CBS-YEAST CBS-DIC CBS-HUMAN CBS-RAT CBS-YEAST CBS-DIC CBS-HUMAN CBS-RAT CBS-YEAST CBS-DIC CBS—HUMAN CBS-RAT CBS-YEAST CBS-DIC CBS-HUMAN CBS—RAT CBS-YEAST CBS-DIC CBS-HUMAN CBS-RAT CBS—YEAST MPSETPQAEV .PSGTSQCED #ooo KCTW..TPNT RCTWQLGRPA RCTWQLGRPM oooooooooo OOOOO LECELVAKCE LKCELLAKCE LKCELLAKCE IKPQIYAKLE 0.00 0.000.. 0' TGIGLALTAA TGIGLALAAA TGIGLALAAA TGIGLALIGA oooo#ooo # PESHIGVAKK PESHVGVAWR PESHVGVAWR PESHIGVAKK oo#oo#- .GKIDMIVCT .GKLDMLVAS .GKVDMLVAS FDNLRAVVAG #0 o #0 NTNK.SYKIE QTEQTTYEVE QTEQTAYEVE KTDITDYKVE oo#oo oo LCAGSSGSAM LCGGSAGSTV LCGGSSGSAM LVGGSSGSAF oo o VDNGFVDPEY LQKGFLKEED LQKGFMKEE. KKNNLWDDDV GPTGCPHRSG GSAGCPQDLE TENTPHTTRR SESPHHHTAP ADSPHYHTVP ...... MTKS 0000000000 FFNAGGSVKD FFNAGGSVKD FFNAGGSVKD LYNPGGSIKD ##oo# oo # IKGYKMIITL VRGYRCIIVM VKGYRCIIVM IKGYRTIITL 0 0.0000. LNSEIPNSHI LKNEIPNSHI LKNEIPNSHI LEKEIPGAVI 0.0.0.0.. AGTGGTITGI VGTGGTITGI AGTGGTITGI AGTGGTISGI 00000000 0 GIGYDFIPNV GIGYDFIPTV GIGYDFIPTV GIGYDFVPQV o# oo VGALLAAK.. AVAVKAAQ.. AVAVKAAQ.. TAVVKYCEDH KTK ....... LTE ....... LSV ....... LARFDSSKLE 1315 PHSAKGSLEK VQPEKGQLEK o #o TPKKLIMDNI AKSPKILPDI TKSPKILPDI EQQADSRHNV O. 0.0 o. RIGHRMIVDA RISLRMIEDA RISLRMIEDA RIAKSMVEEA 00000 0000 PEKMSQEKVD PEKMSSEKVD PEKMSMEKVD PEKMSNEKVS 0000 o 000 LDQYGNPSNP LDQYRNASNP LDQYRNASNP LDQYNNMMNP ooo#o-# o ARKIKERLPN ARKLKEKCPG ARKLKEKCPG SKYLKEQNDK o#o #00 o LERKLVDQWI LDRTVVDKWF LDRAVVDRWF LDRKLIDVWY 0 0000' .QLKKGQRCV .ELQEGQRCV .ELKEGQRCV PELTEDDVIV ..DQQEEEKY ..KKP...WW ..KRP...WW ASTTKYADVF GSPEDKEAKE GASGD...KE o oo ..#o LDNIGGTPLV LKKIGDTPMV LRKIGNTPMV IDLVGNTPLI #o o EESGRIKKG. ERDGTLKPG. ERAGTLKPG. EASGRIHPSR oo#ooo#oo# VLKALGGEII VLRALGAEIV VLRALGAEIV VLKALGAEII 0.0.0 .0 o LAHYDGTAEE LAHYDTTADE LAHYDDTAEE EAHYFGTGRE O ##0000 o CIVVGVDPHG CRIIGVDPEG CKIIGVDPEG IQIVGADPFG .# O O KTDDKESFIM KSNDEEAFTF KSNDDDSFAF KTDDKPSFKY o#o ..#.o. VLLADSIRNY VILPDSVRNY VILPDSVRNY AIFPDSIRSY o##o HGATVKDLTL WHLRVQELGL WHLRVQELSP GNATVKDLHL 08 oo ..MSAPEGPS PLWIRPDAPS RVWISPDTPS oooooooooo o#o# a # RVNKVSS..D RINKIGKKFG RINRISKNAG ALKKLPKALG Qo#ooooooo DTLIEPTSGN DTIIEPTSGN DTIIEPTSGN STLIEPTSGN 0000 o O. RTPTEAAFDA RTPTNARFDS RTPTNARFDS RTPTAAAWDS oo#oo# LLEQCE.... ILQQCD.... ILQQCD.... IQRQLEDLNL SILAQPESLN SILAEPEELN SILAEPEELN SILAQPENLN oo# ARRLIKEEGL ARMLIAQEGL ARMLISQEGL ARQLISNEGV o . o . -# MTKHLNDDWL MTKFLSDRWM MSKFLSDKWM LTKFVDDEWL o#o# . PKPITISATT SAPLTVLPTI SAPLTVLPTV KPVVSVKETA CBS-DIC CBS-HUMAN CBS-RAT CBS—YEAST CBS-DIC CBS-HUMAN CBS~RAT CBS-YEAST CBS-DIC CBS-HUMAN CBS-RAT CBS-YEAST oo # #-# TCAAAVQLLQ TCGHTIEILR TCEHTIAILR KVTDVIKILK ....... KKA ....... GKV ....... GKV KGKYLDFKKL o#o AIVTEND... ALVVHEQIQY ALVVHEQIQY AVITDG.... 0000 000 QYGFDQLPVV EKGFDQAPVV EKGFDQAPVV DNGFDQLPVL o o o 00 VPTDAVSKVM QPSDQVGKVI RPSDEVCKVL NNFNDVSSYN HSTGKSSQRQ RNNGVSSKQL ......... L 1346 . #o SESKKVLVNS DEAGVILGMV NESGAILGMV TEDGKLSGLV FRFTKNEKYI YK ..... QFK YK ..... QFK ENKSGKKKFI #0. o. EIISIVTKID MVFGVVTAID MVFGVVTAID KPIHIVTKMD o o LLVTSL.TYA TLGNMLSSLL TLGNMLSSLL TLSELLRKLS O o PITQSTSLAT QIRLTDTLGR PIHLTDTLGM KFDENSKLSD ## LLTYLMKSQQ LLNFVAAQER LLNFVAAREQ LLSYLA.... S ......... A ......... A ......... INNSNNDNTI .0 O LSKFFENHSS LSHILEMDHF LSHILEMDHF LNRFFEKNSS KN. DQK TRK 147 enzyme fragment suggesting that the gene for DdCBS was present as a single copy( Fig.3). (C) Northern blot analysis Northern blot analysis was done as previously described (Hassanain and Kopachik, 1989). DdCBS mRNA level was highest in the actively growing vegetative stage and gradually reduced in developmental stages (Fig.4A). Some of the expression of developmentally regulated genes in this organism is controlled by CAMP (Firtel, 1991). To Check whether DdCBS mRNA level was regulated by CAMP, 50nM CAMP was given every 10 min for 8 h to cells shaking as a suspension in DB buffer. The DdCBS mRNA level between CAMP-pulsed and control cells (without CAMP) showed about a 5 fold reduction by CAMP pulse (Fig.4B). While other mRNAs show different types of regulation by CAMP; Clone Dblp mRNA level had no Change and Clone EHJ-I showed 20 fold reduction. The mRNA level in vegetative (Veg) and cells taken after 12 h (T125) in the absence of CAMP pulses in Clone Dblp and clone EHJ-I was unchanged, whereas the DdCBS mRNA level was Clearly reduced (Fig.4B). This suggests that starvation alone as well as CAMP can reduce DdCBS mRNA level in D. discoideum deve10pment. (d) Conclusion (I) An entire coding sequence for Dictyosteli um cystathionine B- synthase has been reported. The deduced amino acid sequence showed 5 1% identity with human and 48% with yeast homologues. 150 Fig. 4. A. Expression of DdCBS mRNA during development on agar DB plate. RNAs were isolated at Vegetative (Veg), 1h (T 1), 2h (T 2), 6h (T6), 9h (T9), 12h (T12), 15h (T15) and 24h (T24) stages. Northern blot analyses were done as previously described (Hassanain and Kopachik, I989). The constitutive level of Clone Dblp mRNA shows that an equal amount of mRNA was present in each lane. B. Regulation of different types of RNA during development in shaken suspension cultures with or without CAMP pulses. Vegetative amoebae were resuspended at 2x107 cells/ml in DB buffer. After 4hr some cultures received additions of 5011M CAMP every 10min for the next 8hr. Total RNA was isolated from cells without (T12S) and with CAMP pulses (TIZP) in suspension culture. The same set of RNA blots were probed with DdCBS (a), Clone EHJ-l (b), and Clone Dblp (C). 22x1:- 9Kb-' 5th- 149 148 Fig. 3. Southern blot analysis of D. discoideum genomic DNA. 10 p g of DNA was digested by restriction enzymes and separated on 0.7% agarose gel. DNA was transferred to Genescreen (NEN) membrane. Blot was probed with a 32P-labelled Clone 6-1 whole insert or EcoRI/SalI fragment of Clone 6-1. Lane 1: Bgl H, lane 2: EcoR 1, lane 3: Bgl II/EcoR 1, lane 4: Hind IH, lane 5: Hind IH/Pst I were used. 151 A) Veg T1 T2 T6 T9 T12 T15 T24 .----—OOO-Os- DCBS M a... s B) Veg T128 TlZP a. . - o. . DCBS b. .- . Clone V4-7 152 (2) Southern hybridization analysis implied that DdCBS is a single copy gene. (3) DdCBS mRNA level was reduced in developing cells. (4) DdCBS mRNA level was reduced by starvation as well as by CAMP signaling during Dictyosteli um development. ACKNOWIEDGMENTS We thank Dr. Herbert L. Ennis (Roche Institute of Molecular Biology) for providing vegetative AZAP CDNA library and Dr. Neal R. Band for critical comments on manuscript. REFERENCES Byme, C. R., Monroe, R.S., Ward, K.A., and Kredich, N.A.: DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK regions to ptsH. J. Bacteriol. 170 (1988) 3150-3157. Cherest, H., Thomas, D., and Surdin-Kerjan, Y.: Cysteine biosynthesis in Saccharomyces ceresvisiae occurs through the transulfuration pathway which has been built up by enzyme recruitment. J. Bacteriol. 175 (1993) 5366-5374. Devereux, J ., Haebberli, P., and Srnithies, O.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-397. Firtel, R.A.: Signal trasduction pathways controlling multicellular development in Dictyostelium. Trends in Genetics 7 (1991) 381- 388. 153 Griffith, O.W.: Mammalian sulfur amino acid metabolism: an overview. Methods Enzymol. 143 (1987) 366-365. Hassanain, H.H., and Kopachik, W.: Regulatory signals affecting a selective loss of mRNA in Dictyostelium discoideum. J .Cell Sci. 94 (1989) 501-509. Ishihara, S., Morohashi, K., Sadano, H., Kawabata, S., Gotch, O., and Omura, T.: Molecular Cloning and sequence analysis of CDNA coding for rat liver hemoprotein H-450. J. Biochem. 108 (1990) 899-902. Kruger, W.D., and Cox, D.R.: A yeast system for expression of human cystathionine B-synthase: structional and functional conservation of the human and yeast genes. Proc. Natl. Acad. Sci. USA. 91 (1994) 6614-6618. ' Loomis, W.F.: The development of Dictyostelium discoideum. Academic press, New York, NY, 1982. Mudd, S.H., Levy, H.L., and Skovby, F.: In the metabolic basis of inherited disease, eds. Scriver, D., Beaudet, A., Sly, W. and Valle, D. MCGraw-Hill, New York, NY, 1989, pp. 693-734. Sirko, A., Hryniewica, M., Hulanicka, D., and Back, A.: Sulfate and thiosulfate transport in Escherichia coli K12: nucleotide sequence and expression of the cycTWAM gene Cluster. J. Bacteriol. 172 (1990) 3351-3357. Steel, L.F., Smyth, A., and Jacobson, A.: Nucleotide sequence and Characterization of the transcript of Dictyosteli um ribosomal protein gene. Nucleic Acids Res. 15 (1987) 10285-10298. Swaroop, M., Bradley, K., Ohura, T., Tahara, T., Roper, M.D., Rosenberg, LE, and Kraus, J .P.: Rat cystathionine B-synthase. J .Biol. Chem. 267 (1992) 1465-11461. 154 APPENDIX III : MATERIALS AND METHODS Basic molecular Cloning techniques were followed according to “Molecular Cloning (Sambrook et al., 1989). Growth and Differentiation of Dictyas teli um In this work a strain KAX4 from Dr. Kessin was used. 1. To start a culture of axenic cells spread a suspension of Klebsiella pneumoniae in SW2 broth onto 0.1 LP plate. Spread some of the silica gel crystals which contain spores onto the plate and incubate at I9-22°C. Place the plates right side up (lid on top) in a plastic box and cover. 2. When a plate of fruiting bodies is available, collect spores from the fruiting bodies with inoculation loop. Transfer spores into HL—5 broth containing 200 pg/ ml streptomycin sulfate and 10 ug/ ml penicillin G in 15 ml tube. The final concentration of spores should be more than 3 x 107/ml. 3. Place the tube in the incubator and shake at 150 rpm for about 3-4 days then count the amoebae. When the concentration reaches more than 8 x 106/ ml dilute to 2 ml and then allow to grow to that concentration again in about a day. Next day dilute cells to 1 x 105ml in 50 ml flask containing 10 ml of HL—5 media. Shake the cells at 150 rpm in the incubator and subculture every day or every the other day. 4. T a harvest cells for suspension development experiments spin the cells down at 1,500 rpm in the table top centrifuge for 2 min. 155 Wash the pellet with equal amount of Sorensen's buffer or KK2 buffer and spin down again. Resuspend cells in a buffer at 5 x 106 to 2 x 107/ ml and then shake cells at 150 rpm in the incubator. 5. Cells are placed on non-nutrient agar (NNA) for Checking normal development. To do this spread 1-2 x 107 washed cells out evenly on NNA buffered with KK2 or DB or SB. Adjust the wettness of the plates by adding buffer if too dry or drying with the lid off to evaporate same buffer if too wet. Then place in the incubator. MEDIA AND BUFFERS a. SB : Sorensen's phosphate buffer 17 mM pH 6.0. Prepare a 1 M stock solution: KHZPO4 120.33 g NaIHPO4 17.92 g Nana pure water to 1 L (Check pH and adjust to 6.0) Autoclave 25 min. b. DB : Development buffer, Sodium phosphate 10 mM pH 6.5. Prepare a 1 M stock solution of phosphate buffer: NaZHPO4 45.44 g NaHZPO4 93.84 g NaOH pellets 9.0 g Nana pure water to 1 L ( Check pH and adjust to 6.5) Autoclave 25 min. Add 10 ml of stock and 0.2 ml of 1 M sterile CaCl2 and 2 ml of 1 M MgSO4 to nano pure water to make 1 L. Store at RT. 156 C. KK2 : Potassium phosphate buffer, 40 mM pH 6.4. Prepare a 10 X stock: To KHZPO4 (54.4g) add about 800 ml of nano pure water and about 155 ml of 1 M KOH and pH to 6.4. Adjust to 1 L. Autoclave 25 min. Dilute 1 : 10 with nano pure water to make a 1X solution. (1. SM/Z : Rich nutrient media in plates or as a broth. Bacto peptone 5 g Yeast extract 0.5 g Glucose 5 g MgSO4 X 7 H20 1 g KHZPO4 2.2 g NaZHPO4 1.0 g d water to 1000 ml Add BBL or Bacto agar 20 g per liter for plates. Autoclave 40 min. e. 0.1 LP : Weak nutrient media for plates Agar 15 g Lactose 1 g Bacto peptone 1 g KHZPO4 2.05 g NaZHPO4 0.03 g d water to 1000 ml Autoclave 40 min. f. HL—5 : AxeniC broth for KAX4. Glucose 14 g Add 100 ml water in flask. Yeast extract 7 g BBL Thiotone 14 g Add 600 ml water in bottle. KHZPO4 0.5 g 157 NaHZPO4 0.5 g Add 100 ml water in flask. Autoclave all three solution 40 min. After autoclaving combine the solutions and make to 1000 ml. Add 1 ml of 1 M HCl to adjust pH to 6.5. g. Streptomycin sulfate. Prepare a stock solution of 20 mg/ ml. Weigh out and dissolve then filter through a 0.45 pm Millipore filter and store at -20°C. h. Penicillin G. Prepare a stock solution of 100 mg/ ml as for ‘g’ above. Differential Display PCR 1. First strand CDNA synthesis 5 X Reaction buffer : 4 111 RNasin ( Promega) 0.5111 dNTPmix(100uMeaCh) 1111 DTT ( 100 mM) 1 111 14 mer (25 11M) 2 111 Total RNA( G/SCN prep ) 1.5 111 ( 2-3 pg ) MMLV reverse transcriptase 1.5 111 ( 300 U ) Diethylpyrocarbonate treated (Dep'd) water 7.5 ul Incubate at 37°C for 1 h. After incubation heat at 95°C for 2 min to inactivate enzymes. 2. PCR 158 1 111 of arbitrary 10 mer + 2 111 of the first strand CDNA + 17 ul of PCR labeling mix + 3 units of Taq polymerase. Step I 94°C 30 8 Step 2 42°C 1 min Step 3 72°C 30 5 Repeat 30 cycles. 3. Load 6.5 111 of PCR product on 6 % sequencing gel. Follow the procedure written in "Sequencing gel preparation and electrophoresis". However, to isolate differentially expressed band do not fix the gel. PCR labeling mix : Dep'd water 25.3 111 10 X Reaction buffer 5 111 dNTPs (250 11M each) 4 111 14 mer (50 11M) 2.5 111 10 mer (10 M) 2.5 ul MgCl2 (25 mM) 2.8 ul 355-dATP 2.8 1111 Recovery and Reamplification of DNA from sequencing gel DNA should be amplified to subclone into a plasmid. 159 1. Cut differentially expressed DNA band from sequencing gel and then hydrate in 140 pl of water and boil 15 min. 2. Spin down and transfer supernatant then add NaAC to 0.3 M with 0.4 pl of linear polyacrylamide (5 mg/ ml). Add 2 volume of EtOH. Keep in -70°C at least 30 min. Spin down and wash the pellet with 70 % EtOH. Dry pellet and dissolve in 10 pl water. 3. PCR HZO 17.9 pl 10 X buffer 4.0 pl dNTPs (250 pM each) 3.2 pl 14 mer (50 pM) 2.0 pl 10 mer (10 pM) 2.0 pl EtOH ppt. DNA 8.0 pl MgCl2 (25 mM) 2.4 pl Taq Pol (Promega) 0.5 pl After 40 cycles (94°C, 30 s; 40°C, 2 min; 72°C, 30 8) add 0.5 pl of Taq Polymerase and do PCR 15 more cycles. link 5 min final extension at 72°C. Run agarose gel and isolate insert by using QLAEX Gel Extraction Kit (QIAGEN) and dissolve in 20 pl of TE. 4. Iigate the amplified insert DNA into pCRII vector (TA Cloning Kit, Invitrogen). Radioactive Probe Preparation (Feinberg and Vogelstein, et al., 1983) - Random primer method (Oligolabelling) Note: TM: 01.: LS: 160 Dilute 100-200 ng of purified DNA fragment to 12 pl in dHZO. Boil for 10 min to denature and then quick-cool in ice- water for 2 min. Add following: LS 18 pl BSA 1 pl (16 mg/ml) dNTPs 3 111 (dGTP, dATP & dTTP) {32deCTP 5 pl (50 pCi) Klenow frag. 8 unit (DNA polymerase 1) total 40 pl Incubate at RT for 5 h to overnight 250 mM Tris.Cl pH 8.0 2.5 ml 25 mM MgCl2 0.25 ml 50 mM B-Mercaptoethanol 36 pl Water to total 10 ml 90 OD. Units/ ml Hexamers 1 mM Tris. Cl pH 8.0 1 mM EDTA pH 8.0 1 MHepes (pH 6.6) : TM : OL, 25 : 25 : 7 Screening of A CDNA library 1. Preparation of blot. a. Grow LE 392 cells in LB containing maltose and MgCl2 overnight. 161 100mlLB+ 1 mlof20%maltose+ lmlofl MMgCl2 b. Add 0.1 ml of culture to fresh media and grow several h. C. Spin down culture in the table top centrifuge at maximum speed for 5 min. (1. Pour off supernatant and resuspend cells in half volume of SM buffer. e. Add 0.1 H11 LE3 92 cells into 5 ml glass tube. f. Add 2 pl of 1000 fold diluted A ZAP CDNA library to LE3 92 cells. g. Incubate at 37°C for 30 min. h. Add 3 ml of top agar. i. Plate out on LB agar and incubate at 37°C for 4-6 h. j. After plaques are formed leave the plate in the refrigerator for 1 h. k. Use NEN Colony/PlaqueScreen membrane and follow the manufacture's protocol. To avoid false positives make two blots from a plate. 2. Screening the blot Use same buffer as used for Southern blot and follow same procedure which was described in Southern blot. 3. Identification of positive and re-screen blot. a. Find the location of positive plaques by overlapping two X-ray films and the plate. b. Isolate agar plug from the plate by wide side of pasteur pipet. C. Transfer agar plug in 1 ml of SM and leave at RT for 1 h. (1. While incubating prepare infectable LE392 cells as described in "Preparation of blot". 162 e. Add 2 pl or 20 pl of 'C' solution into LE392 cells. f. Follow same procedure from step lg to 2. g. Isolate single positive plaque by overlapping the plate and X-ray film. h. To excise plasmid DNA from A ZAP Clone follow the procedure written in "Excision of Plasmids from the A ZAP CDNA Clone". SM : NaCl 2.9 g MgSO4 1.0 g I M Tris 25 ml (pH 7.5) 2 % Gelatin solution 2.5 ml H20 to 500 ml A Top agarose Tryptone 2.0 g NaCl 1.0 g Agarose 1.6 g HZO to 200 ml Autoclave then add 2 ml of 1 M MgSO4 + 2 ml of 2 % maltose after cool. Excision of Plasmids from the A ZAP CDNA Clone For sequencing and other manipulation of DNA it is required to isolate plasmid DNA from A ZAP CDNA Clone. 163 1. Pick out positive plaque and dissolve in 50 p1 of SM buffer. Leave it in the refrigerator overnight. 2. Mix 200 pl of ODz1.0 SURE cells, 10 pl of A ZAP phage stock, 190 pl of SM buffer, and 20 pl of R408 helper phage in 15 ml test tube. Incubate this mixture at 37°C for 15 min. 3. Add 5 ml of 2X YT medium (10 g NaCl, 10 g yeast extract, 16 g tryptone per liter of H20) and incubate at 37°C for 4 to 6 h with shaking. 4. After incubation, heat the mixture to 70°C for 20 min to inactivate the present A phage and to kill the bacteria. 5. Spin down cells for 5 min at 2,000 rpm in table top centrifuge. 6. Save supernatant which contains the Bluescript phagemid packaged in the f1 phage particle, as well as the f1 helper phages. 7. To recover the excised phagemid from this stock, mix 200 pl of OD=1.0 DHS aF' host cells with 200 pl of phagemid stock and then incubate at 37°C for 15 min. 8. Spread 200-300 pl to LB-amp plate with 50 pl of 2% X-gal and 10 pl of IPTG. 9. Select white colonies and Check insert. A ZAP CDNA library DNA isolation A ZAP CDNA library DNA was used for anchored PCR. 1. Prepare infectable LE3 92 cells as described in "Screening of A CDNA library". Add 1-2 111 of A ZAP CDNA library (titer 1010/ml ) t0 1 ml of fresh LE392 cells. Incubate the tubes at 37°C glass bead bath 164 for 30 min and transfer into 100-200 ml of LB-Mg++ (LB + maltose and magnesium). 2. Shake in the 37°C incubator for 5-7 h. It should be Cloudy in lb and Clear upon lysis in 5-7 h. 3. Spin down with 8000 g at 4°C for 10 min. A is in the supernatant. 4. Add RNase A to 1 pg/ml and 50 pl of crude DNase to the supernatant and incubate at 37°C for 30 min. 5. Add 5.8 g of NaCl. Swirl to dissolve. Keep on ice for 1 h. 6. Spin down with 11,000 g at 4°C for 10 min. Keep the supernatant. 7. Add PEG (8000) to 10 %. Stir slowly at RT then keep on ice for at least 1 h or overnight. 8. Spin doWn with 11,000 g at 4°C for 10 min. Drain throughly and leave the tube inverted for 5 min or more. 9. Resuspend phage in 3-4 ml SM. 10. Add equal volume of CHCl3 and vortex 30 s. Spin down at maxrnum speed in the table top centrifuge for 15 min.Place aqueous phase on the CsCl step gradient after dissolving 0.5 g CsCl per ml. Step gradient in 17 ml polyallomer tube. 3 ml 1.7 g/ml 2 ml 1.5 g/ml 2 ml 1.45 g/ ml Spin AH-629 with 22 K rpm at 4°C for 2 h. 1 1. Hold the tube in stand. Place light above and use dark paper or board for background if blue band is not easy to see. 165 12. Use tape over tube and puncture with 21 gauge needle in 1 ml syringe. Pull out 0.5-1 ml. 13. Dialyze against 1 L of 10 mM NaCl/ 50 mM Tris pH 8/ 10 mM MgCl2 for 1 h then replace with fresh buffer and dialyze one more h in the cold room. 14. Collect dialysate and add RNase A to 50 pg/ ml. Incubate at 37°C for 30 min. 15. Add EDTA pH8 to 20 mM and proteinase K to 50 pg/rnl and SDS (Serva SDS) to 0.5%. Gently mix and keep on 55°C water bath for 1 h. 16. Cool. Then extract with phenol, phenol: CHCl3 (50:50) and CHC13. 17. Add 3 M NaAC (pH 7.0 not 5.0) to 0.3 M and mix. Add 2 volumes of EtOH. Invert tube to mix and watch for thin thread of DNA. Remove with pipette tip. Redissolve without vigorous mixing in 100 pl of TE. 18. Check yield on agarose gel. If there is too much RNA present, add RNase A to 100 pg/ml. Incubate at 37°C for 30 min to 1 h. 19. Extract RNaseA with Phenol/ chloroform and chloroform. 20. Spin through mini G-50 column in STE. See "Plasmid isolation" for directions. EtOH precipitation. Dissolve DNA in 100 pl TE. Anchored PCR To get 5'end CDNA of A ZAP Clone sequence anchored PCR was done by using A ZAP CDNA library DNA as a template. 166 A ZAP CDNA (10 ng) 1 pl 10 X buffer 10 pl MgCl2 (25 mM) 6 pl dNTPs (250pM each) 8 pl BS-SK primer (50 pM) 2 pl Gene specific primer (50 pM) 2 pl H20 72 pl PCR 35 cycles (94°C, 1 min; 55°C, 1.5 min; 72°C, 3 min). link 15 min final extension at 72°C. Run agarose gel and isolate insert by using QIAEX Gel Extraction Kit (QIAGEN) and dissolve in 20 pl of TE. Iigate the amplified insert DNA into pCRII vector (TA Cloning Kit, Invitrogen). Plasmid Isolation I. Mini-preparation methods A. Alkaline lysis method I. Grow 5 ml of isolated Clone in LB or SOB containing 100 pg/ ml ampicillin. Shake rapidly in the 37°C incubator at least 9 h until stationary phase. 2. Spin down cells in microfuge 1 min. 167 3. Resuspend pellet with freshly prepared 100 pl of 50 mM glucose/ 25 mM Tris (pH 8) / 10 mM EDTA/ 2 mg/ml lysozyme. Ice 10 min. 4. Add 200 pl 0.2N NaOH/ 1% SDS and mix gently to lysis cells. Do not vortex. Prepare fresh NaOH/ SDS solution. Ice 10 min. 5. Add 150 pl of 3 M sodium acetate (pH 5) and mix by inversion. Leave in -20°C freezer for 10 min. 6. Spin in SH-MT rotor at 13,500 rpm for 10 min at 20°C or maximum speed in microfuge. Recover the supernatant and add 2 volumes of cold EtOH. Precipitate at -20°C or -70°C freezer at least 10 min. 7. Spin in SH—MT rotor for 10 min. Aspirate off supernatant. Wash the pellet with 70% cold EtOH. Dry the pellet in the Speed Vac. 8. Resuspend pellet with TE which contains 100 pg/ ml RNAse A. Incubate for 30 min at 37°C. 9. Add equal volume of 50 : 50 phenol/ chloroform (pH8). Vortex 1 min and spin down 5 min in microfuge. Repeat extraction with one quarter volume of chloroform alone. Collect supernatant (aqueous phase). 10. Prepare mini-G50 column. Add G50 Sephadex equilibrated with 0.1M NaCl in TE pH 8.0 to spin column tubes only up to the neck. Place tube inside of larger collection tube. Spin in microfuge for 1 min. Check G50 level and spin down again for 5 min. Place the G50 column in a new tube then add aqueous phase to G50 and spin for 5 min. 168 1 1. Add 2 volumes of EtOH. Precipitate DNA for at least 10 min in -70°C freezer. Spin down and dry as before. Resuspend DNA in 40 pl of TE or water. B. Speedprep ( Goode and Feinstein, 1992) This method is good for Checking insert. 1. Spin down 1.5 ml of overnight cultured bacteria. 2. Aspirate supernatant and resuspend pellet in 100 pl of solution A (50 mM Tris (pH8.0), 4 % Triton X-100, 2.5 M IiCl, 62.5 mM EDTA). 3. Add 100 pl of a Tris-buffered phenol/ chloroform mixture (1:1). Vortex the tube for 10 s and microfuge at top speed for 2 min. 4. Remove the plasmid-containing 200 pl of cold 100% EtOH. Vortex the sample briefly, and microfuge it at top speed for 5 min. 5. Discard the supernatant. Wash the pellet with 1 ml of 70% EtOH and dry in Speed Vac. 6. Resuspend the pellet in 10 p1 of TE which contains 100 pg/ ml RNAse A. Incubate at RT for 5 min. This DNA can be used for restriction enzyme digestion. C. Wizard miniprep (Promega) After the presence of insert is confirmed by Speedprep method Wizard miniprep kit was used to isolate plasmid for sequencing. The manufacture's protocol was followed. 11. Large Scale Plasmid Isolation A. Cesium Chloride Method 169 I. Grow to saturation 100 ml of cells in LB or SOB with 100 pg/ ml arnpicilin. 2. Spin down cells in HB-4 or GSA rotor at 7 K for 5 min. Resuspend the pellet of cells in 2 ml of sucrose buffer (25 % sucrose/ 50 mM Tris pH 8/ 10 mM EDTA). Pipet up and down and vortex to resuspend all cells. Transfer to a Clear T865 tube. 3. Add 0.6 ml of 5 mg/ ml lysozyme in 50 mM Tris/ 10 mM EDTA. Keep on ice for 5 min. 4. Add 1.2 ml of Tris/ EDTA the 50 pl of 10 mg/ ml boiled RNAse A. Keep on ice for 5 min. 5. Mix to resuspend cells then slowly add with swirling 5 ml of 2 % triton/ 50 mM Tris/ 10 mM EDTA. Leave 10 min at RT. 6. Spin in the ultracentrifuge T865 rotoer for 20 min at 25K at 20°C. The pellet should be slightly fluffy at the top but doesn't have to be. 7. Pour off supernatant into a 15 ml tube and make to 10 ml with 0.4 ml of 5 mg/ ml EtBr. Dissolve 9.6 g of CsCl in the solution by warming at 55°C and shaking for 15 min. Spin the tube at maximum speed in the table top centrifuge for 10 min. Decant the solution under the red protein floating on top. Load the sample into two TV865 tubes and spin at 50 K for at least 8 h at 20°C with the reograde mode. 8. Gently pick up rotor and take tubes into the darkroom. Identify the red band with the 366 nm uv lamp and collect DNA with 20 gauge needle and one ml syringe. 170 9. Extract the EtBr by adding an equal volume of isopropyl alcohol saturated with 5 M NaCl. Repeat several times until the pink color is gone. 10. Dialyze in 10 mM Tris/ 1 mM EDTA buffer at least 6 h. 1 1. Extract dialysate with equal volume of 50:50 phenol/ chloroform buffered with 0.1 M Tris pH 8. 12. EtOH precipitation. Add 0.1 volume of 3 M sodium acetate pH 5 and two volumes of cold EtOH. Spin down at 13 K for 10 min and wash the pellet with 70% EtOH. Dry the pellet and resuspend with 200 pl of TE. B. Qiagen Maxi Plasmid Kit. Follow the manufacture's protocol. Transformation of Dictyostelium W. Nellen et al., in Methods in Cell Biology voulrne 28; 67-100, Spudich ed. (1987) was a general reference for an early version of transformation. 1. The Kessin strain of AX3 (KAX4) was used for transformation experiment. Cells within a week or two were used. During growth keep the cell concentration in early log phase (1. e. below 2 x 10" cells/ml). 2. Dispense 10 ml containing 1 x 107 cells onto a 100 mm tissue culture plate. Allow cells to settle out of solution at least 1 h. 3. Remove the HL-5 media and replace with 10 ml of MES/ HL-5 pH 6.3. Tilt plate and pipette off from one marked edge of plate. 171 Make all subsequent additions from this one area to prevent too much cell loss from pipetting steps. Leave at least 30 min for cells to resettle. 4. Prepare DNA (12 pg) for addition. To 0.6 ml of HBS add 38 pl of 1 M CaCl2 and the DNA while vortexing. Allow to precipitate for 30 min. 5. Pipet off media and then vortex DNA tube to resuspend DNA and dump by drops in the middle of the plate. Rock gently to spread the DNA and leave for 30 min. 6. Add 10 ml of MES/HL-S pH 6.3 and let stand for 3-5 h. 7. Pipet off media and add 2 ml of 18% glycerol in HBS. Let stand for 2 min and then pipet off residual glycerol. Quickly add 10 ml of HL-S. Let stand overnight (12-18 h). 8. Pipet off media and add 10 ml of HL—5 plus 20—40 pg/ ml G418. Let stand for 2 days. Change media with drug every two or three more days. 9. Colonies become visible as faint patches when the plate is held up to the light and tilted. Colonies with the endogenous plasmid (aneI or aneAI) becomes visible in about 5 days but colonies with an integrating plasmid (pDneoII, BIOS) become visible in 12 to 18 days. 10. When faint patches of colonies visible Change media with drug to remove floating cells. Pipet off cells from the middle of patch area by using P-200 pipetman. Transfer those cells to new 100 mm tissue culture plate which contains media with G418. It will take about 3 days to form patches of colonies from isolated Clones. 172 1 1. When the colonies have become almost confluent or at least covering one half of the plate wash the cells off the plate and into a 10 ml flask for suspension growth with drug selection. Notes. - Sterilize DNA by heating in the heating block at 70°C for several hours. - Sterilize G418 is prepared as 4 mg/ ml water by filtration. Make 1 ml aliquots and store at -20°C. - Sterilize MES buffer (1 M stock) by filtration. MES is not stable to autoclaving. - 18 % glycerol : To 3 ml of 60 % ultrapure sterile glycerol add 5 ml of 2 x HBS and 2 ml of water. -HBS is prepared as a 2 x concentrate and stored frozen. 4 g NaCl 0.18 g KCI 0.05 g NaZHPO, or 0.062 g NaZHPO4/ H20 2.5 g HEPES 0.5 g Dextrose pH to 7.05 with NaOH (proper pH is important) nanopure water to 250 ml. Filter sterilize and store as 50 ml aliquots in -20°C. - Sterilize 2 M CaCl2 and stored at -20°C. Genomic DNA isolation 173 I. Grow 4 L of KAX4 cells in HL-S until cell concentration reaches to 0.9-1.1 x 107. 2. Harvest cells. a. Centrifuge cells at 1500 rpm, 0°C for 5 min. b. Resuspend the pellet with 500 n11 of ice-cold 17 mM SB buffer. Spin down cells as above. C. Repeat step ‘b’. 3. Preparation of nuclei. 3. Add 100 ml of nuclei buffer to the pellet. b. Add 0.65 ml of 100% NP-40 and shake vigorously about 5 min. Check lysis with microscope. C. If cells are completely lysed centrifuge lysate 8000 rpm at 10°C for 10 min to pellet nuclei. d. Resuspend nuclei pellet with 100 ml nuclei buffer. e. Add 0.55 ml of 100% NP-40 and shake vigorously to remove mitochondria. _ f. Spin down lysate with 8000 rpm at 10°C for 10 min 4. Isolation of nuclear DNA a. Resuspend nuclei pellet with 30 ml of 0.1 M EDTA. b. Add 3.5 ml of 20 % N-lauroyl sarcosine by dripping. C. Incubate at 65°C for 15 min. (1. Transfer that into 50 ml of yellow cap tube. e. Add 2.8 ml of 5 mg/ ml EtBr and CsCl2 to final density of 1.55 g/ ml. f. After dissolving centrifuge the tube at maximum speed in the tabe top centrifuge for 10 min to remove proteins. 174 g. Transfer the Clear solution to T865 tube and centrifuge with 35K rpm in 20°C for at least 36 h. h. Isolate DNA band by using 21 gauge needle and Iml syringe. i. Extract EtBr by adding an equal volume of isopropyl alcohol saturated with 5 M NaCl. Repeat several times until the pink color is gone. j. Dialyze in 10 mM Tris/1 mM EDTA buffer at least 6 h. k. Collect dialysate and add 2 volume of cold EtOH and 1/ 40 volume of 4 M NaCl. Spool out DNA by using yellow tip. . l. Dissolve in 1 ml of TE. Use shaker for 1 day to dissolve genomic DNA. Nuclei buffer : 0.025 M Tris-HCl, pH 9.2 5 mM MgAC 5 mM EDTA 5 % sucrose Bacterial Cell Transformation I. Grow DH5 a. Pick out about 5 colonies from an LB plate and grow in 5 ml of SOB at 37°C overnight or for at least few h until Cloudy. 175 2. Transfer 100 pl of overnight culture or 0.5 ml of younger culture to 100 ml of SOB in a 500 ml flask. Grow to OD 550 of about 0.5 ( in about 2 h). 3. Cool on ice 10 min. Spin down cells with 7000 rpm at 4°C for 5 min. Resuspend cells in 10 ml of be I. Keep on ice 10 min. 4. Spin as above. Resuspend cells in 2 ml of be 11. Add 70 pl of DMSO. Keep on ice 10 min. 5. Add 70 pl of DMSO and make 200 pl of aliquots in tube and freeze at -70°C. Keep in -70°C until it is needed for transformation. 6. Thaw frozen competent cells on ice and transfer to Falcon 2059 tube. 7. Add about 10 ng of DNA to the tube and keep on ice for 30 min. 8. Heat shock for 50 s at 42°C and quickly transfer to ice again. Keep on ice for 2 min. 9. Add 0.9 ml of SOC and shake the cells in the 37°C incubator for 2h. 10. For blue-white selection spread out 50 pl of 2% X-gal and 10 pl of 0.1 M IPTG on LB-amp plate. When pate is dry, spread out about 50 to 200 pl of culture from 9. Incubate the plates in the 37°C incubator. SOC : To SOB add 18.6 ml of filter sterile 20 % glucose per liter. be I (Transformation buffer 1) 10mMMES pH6.2‘ ImlofIM 100 ml RbClz 1.9 g 45 mM MnCI2 x 4 H20 0.89 g 176 10mMCaCl2 x2H20 1 mlof 1M 3 mM Hexamine cobalt 0.08 g nanopure water to 100 ml then filter sterile and store at 4°C. be II (Transformation buffer 11) 10mMKAC ImlofIMpH7.0 100mMKCl 10mlof1M 45 mM MnCl2 0.89 g 10mMCaCl2 lmlofIM 3 mM HACoCl3 0.08 g 10 % ultrapure glycerol 10 ml nanopure water to 100 ml the filter and store at 4°C. Southern Blot 1. Completely digest 10 pg of genomic DNA in a total reaction volume of 100 pl with Spl restriction enzyme at 37°C for 3 h or overnight. Use only a cut off pipette tip with a wider bore so the DNA is not sheared excessively. Mix the reaction well by pipetting and flicking the tube because the high MW DNA is very viscous. 2. Prepare 0.7% agarose gel with 1 X TBE buffer. Need 400 ml of gel in the 20 X 30 cm gel box and 1.6 L running buffer. Use the large comb slots (0.9 cm X 1.5 cm) or else the DNA may smear during the run. Cover the gel with a thin (2 mm) layer of buffer. 3. Add 10 pl loading buffer to the sample, mix well. Then load the sample in the slots and use lambda DNA (lpg) cut with EcoR I or 177 Hind H1 or both as a size marker at the ends of the gel. Start electrophoresis with EC 500 at 80 V for 10 min to make all of the DNA stack in slot. Then lower voltage to 40 V and electrophoresis overnight for about 15 h until the dye reaches about 10 cm. Perform electrophoresis the same way so that blots done on different days could be easily compared later. 4. Shut off power and cut away the excess gel. Transfer the DNA in gel using 3 X-ray flm as a supporter because the gel breaks easily. Cut off a small bit of one edge of gel as an orientation mark. Place in a large plastic boxs and add about 1 L of buffer then about 2 to 3 drops of EtBr until the solution is just pink. Shake for about 15 min or until the DNA is stained. View on the transillurninator and take a picture with a ruler set along the side of the gel. At the same time, turn on the Haake Cooler set at -10°C. 5. Replace the buffer with 1 L of 0.2 N NaOH /0.6 M NaCl and shake for 30 min to denature the DNA. Replace with I L of 25 mM phosphate buffer (pH 6.5). Change buffer twice within about 30 min. 6. Pour 5 L of phospate buffer into the electoblot apparatus. Cut 4 sheets of 3 MM filter paper and 1 sheet of Gene Screen membrane to the size of the gel to be blotted. Soak the Gene Screen for about 10 min. Assemble the blot sandwhich: on the plastic grid in a box with 3 L of phosphate buffer place a layer of foam, 2 layers of prewetted filter paper, the gel, the prewetted Gene Screen membrane, 2 layers of prewettd filter paper, foam and then the other side of the plastic grid. Do all of assembling process under the buffer to prevent air bubbles from forming in the sandwhich. DNA will not be transfered to the membrane in regions containing air 178 bubbles. Clamp the sandwhich with hands and do not release grip until the sandwhich is within the electroblotter slots and submerged into the buffer. 7. Electroblot with z0.4 Amp for 1 h. Check after 10 min. to make sure the current is stable. Increase current to zI Amp and continue for 6 h or longer. At the end remove the filter and dry on a filter paper. View the blot with the short wave UV light and mark lightly with pencil any lambda size bands or rDNA bands. 8. Fix the DNA on the blot by baking 80°C for 2-4 h. 9. Seal the blot in a seal-a-meal bag with 1 X 10 ml hybridization buffer. Prehybridize the blot at 65°C for at least 1 hr using the agitator set for slow movement of the buffer over the blot. 10. Cut the bag at a corner and add a very hot inset probe at s 10 ng/ m. Re-seal and incubate at the 65°C shaker for at least 2 day. 1 1. For the post-hybridization, wash the blot as follows: a. 2 X 2X SSC RT for 5 min each b. 2 X 0.5X SSC/ 1% SDS at 65°C for 30 min. Check background count (cpm) over region which should not have any counts; if less than 50 cpm, stop here. If the counts still high: C. 1 X 0.2X SSC/ 1% SDS at 65°C for 30 min. Check counts (1. 1 X 0.1X SSC/ 1% SDS at 65°C for 30 min. (There should be almost no detectable Cpm even over the DNA which should contain radiolabelled probe DNA.) Wash away excess SDS with a couple of rinses in 0.1X SSC 179 12. Wrap in plastic wrap and mount over 3 MM filter paper using tape. Mark all size markers, rDNA and any other bands you saw on the blot with radioactive (355) ink. Put date, oreintation marks as X on the filter. Do not get outside wet or else the film in contact will be ruined. Set up blot against XAR-S Kodak film and lay this over an intensifying screen in a cassette. Expose at -70°C for overnight or longer. 13. Keep blot for reuse as many as three times. For reuse, follow procedure for stripping old probe (0.1X SSC/ 1%SDS, boil 30 min). Store the dry blot at RT in plastic bag. S l ' n l. Phosphate buffer for 1 M stock. - Mix 93.84 g NaHPO4 and 45.44 g NaZPO4 in 700 ml water, heat and stir then cool to RT; adjust pH to 6.5 and volume to 1 L. 2. Pre-hybridization buffer: NaCl 2.92g 1 M tris pH 7.5 2.5 ml 50% dextran sulfate 10 ml 50X Denhardt’s solution 5 ml 10% SDS (ultra-pure) 5 ml 10 mg/ ml Salmon DNA 0.5 ml water to total 50 ml RNA Isolation (Chirgwin, et al., 1979) A. Isolation of total cellular RNA from D. discoideum by using guanidinium thiocyanate 180 DALI 1. For each 108 cells (about 1-2 ml cell pellet from 100 ml shaking culture) dissolve in 5 to 10 ml of 4M guanidinium thiocyanate / 0.1 M B-mercaptoethanol/ pH 5. Freeze sample in dry ice or place in -70°C freezer to make sure that all cells lyse. Samples can be stored indefinitely when frozen. 2. Thaw sample then spin at 3000 rpm in a table top centrifuge to pellet insoluble material if any. Centrifugation for 15 to 20 minutes is usually enough. 3. Take an RNAse free AH-629 polyallomer tube and add 2.5 ml of 5.7M CsCl/0.1 M EDTA/ pH 5 to tube to make a CsCl cushion. Gently layer the sample over the cushion. Fill the tube up to within a couple of millimeters of the top. The sample can be up to 13 ml. Balance the tubes to within 0.1g and load all of the buckets even if you only 2 have samples. Centrifuge for 24 h at 25K at 15°C. For the Beckman SW 41 use 2.2 ml of CsCl and spin at 33K and for the SW 28 add 5.5 ml of CsCl and spin at 27 K. Shorter spin times (18 h) could be used without signifcant loss of RNA yield. DAYJ 4. Remove all of the sample by aspiration down to the CsCl layer and then add 3 ml of dep’d water to the tube to wash the walls. Aspirate the wash water and most of the CsCl so that about 1 ml is left. Invert the tube and keep inverted. 5. Carefully add about 0.5 ml of dep’d water to wash the area around the pellet and drain this out. Do not disturb the small 181 button—like translucent pellet of RNA which will be in the exact bottom of the tube. The small aggregates around the pellet are not RNA and should be avoided. They can be wiped out with a Kim-Wipe if necessary. 6. Take up 0.5 ml of dep’d water in a blue tip and jab the pellet to break it up. Add the water and take up the pieces to transfer to a microfuge tube. Add anaother 0.5 ml of water to the cup to rinse and make sure that no RNA is left in the tube or in the blue tip. Now vortex the RNA in the microfuge tube continuously for about 2 min to dissolve the RNA then spin in the microfuge for 5 min to pellet the insoluble material, if any. Take up the supernatant and transfer to a baked Corex 13 ml tube and add 2 ml of dep’d water, 75 pl of 4M NaCl, and 7.5 ml of cold 100% EtOH. Cap with parafilm, invert to mix and store at -20°C for a few hours or overnight or indefinitely. 7. Spin the tube at 13,000 rpm in the HB-4 rotor for 20 min at 4°C to pellet the RNA. Drain or aspirate the supernatant and then gently wash the film of a pellet with cold 80% EtOH using about 0.5 ml to remove the residual NaCl. Remove this wash EtOH by aspirating. Cap the tube with parafilrn and poke holes in it and then place in a vacuum for 5 min or until the EtOH and water have evaporated. The pellet is similar to the appearance of a dry cracked lake bed. Take up the pellet in 200 p1 of dep’d water and vortex to dissolve the RNA. Transfer to a microfuge tube and then add another 200 pl of water to rinse the tube and transfer this to the microfuge 182 tube also. RNA dissolves easily within 2 min of vortexing. If there is pellet material that does not dissolve in this time it is not RNA. This RNA is good enough for use in Northern blots. 8. To further purify the RNA (for in vitro translation and construction of CDNA library) add 0.4 ml of buffered phenol/ chloroform and vortex to make a Cloudy emulsion. Spin for 2 min in the Speedy Vac centrifuge without vacuum to separate the phases. Remove the upper water phase and leave any interface behind. Reextract the water phase in another tube with Choroform/isoamyl alcohol 0.4 ml and spin as before. Remove the lower chloroform phase and reextract with chloroform again. The small amount of residual RNA left in the phenol/ chloroform phase could be recovered by adding 0.2 ml of dep’d water to it and vortexing and spinning as before. If done add this to the ca. 0.3 ml first recovered from chloroform. Add 4M NaCl to 0. 1 M final concentration (12.5 pl to 0.5 ml) and 2 volumes of cold 100% EtOH (1.0 ml to the 0.5 ml). Vortex to mix and allow to precipitate at -20°C for at least 2 hours or overnight or indefinitely. 9. Spin in the microfuge for 10 min and wash the pellet with 50 pl of 80% EtOH. Dryin the Speedy Vac. 10. Add 1 ml of cold 2 M IiCl to the dry pellet and break up the pellet. Cap and rotate or rock for about 1 hour at 4°C. Spin again in the microfuge to recover the pellet and then wash the pellet with 50 ul of 2M LiCl. Remove the wash and add 0.5 ml of dep’d water to dissolve the pellet and then precipitate with NaCl and EtOH as before 183 (step 8). Finally dissolve the RNA in about 100 to 200 pl of dep’d water and determine the OD at 260 to 280 nm. Add 5 pl of the RNA to 1 ml of water and read the ODs. Get at least 0.1 OD units at 260nm, if necessary add more sample. Determine the OD and multiply by the dilution factor of 200 and multiply by 40 pg/ ml to get the RNA concentration in pg/ ml. The usual 260/ 2 80 ratio is about 2 to 2.3 but values as low as 1.6 are still good preparations. 1991115. I. This procedure gives total RNA which can be translated in the reticulocyte assay because inhibitory material has been removed with the extra phenol/ chloroform and IiCl washes. Do not use more than about 2 x 108 cell in the 10 ml at step 1 because a large amount of gelatinous material will spin if the gradient is overloaded. 2. At step 10 the RNA will not dissolve easily occasionally if the RNA concentration is too high and expecially after phenol/ chloroform extraction. These RNA aggregates will need extra help in dissolving such as breaking up the aggregates with a blue tip and vortexing for a long time (over 10 min). Solutions for RNA preparation: 1. All glassware should be baked to be RNAse free (350 °F for at least 3 hours). 2. All plasticware is OK if not handled with bare hands which have RNAse. Wear gloves throughout the procedure. Have pipette 184 tips and microfuge tubes and glassware that you will use only for RNA preparation. 3. Water. Use nanopure water that has been treated with diethypyrocarbonate (dep) at a final concentration of 0.2% as follows. Make a 10% solution of dep in 100% EtOH and dilute this 500 fold in the water. Leave overnight or at least 3 hours and then autoclave for 20 min to inactivate the residual dep. Store the water in small baked bottles. Use gloves when handling the dep because it is a strong denaturing agent. 4. 4M NaCl. Treat with dep as above. 5. Polyallomer tubes. Treat with dep as above. Place the tubes in a large beaker with water and add the dep to 0.2% and mix up. Autoclave as before and store the tubes in a Closed box. 6. Guanidinium thiocyanate/0.1 M B—mercaptoethanol. Add 47.2 g of Fluka G/SCN or ICN or BRL to about 70 ml of nanopure water and then in a hood add 0.7 ml of B—mercaptoethanol. Cover and stir and gently warm to dissolve and make up to a final volume of 100 ml. Filter through a 0.45 pm Nalgene filter and store at -70°C in 50 ml plastic tubes. Check pH, it should be 5 and will be if Fluka G/SCN is used. Adjust the pH if necessary with acid. 7. 5.7 M CsCl/0.1 M EDTA. Add 48 g of baked CsCl technical grade to about 30 ml of nanopure water and then 5 ml of 0.5 M EDTA and stir and warm to dissolve at a final volume of 50 ml. Adjust the pH to 5 with acid. Filter through a Nalgene filter and store at -20 C or add dep to 0.2 % and treat as for dep’d water. 8. Phenol/Chloroform. Shake phenol with 0.1M Tris (pH8) (usually 25ml of phenol with 25ml of Tris) to equilibrate. Spin in 50 185 ml plastic tube at 3000 rpm for 5 min to separate the phases. Repeat until the water phase is pH about 7-8. This may be several times. Add a pinch of 8-hydroxyquinoline to give the phenol some brown color for visibility and then add B-mercaptoethanol to 0.1%. This makes the equilibrated phenol which can be stored at 4°C tightly capped. Add equal parts of this phenol to chloroform to make the 50:50 mixture. 9. Chloroform/isoamyl alcohol. Add isoamyl to make a 50 to one solution of chloroform to isoamyl alcohol and store this at room temperature. B. Isolation of Total RNA by using kit RNA STAT-60 (TEL-TEST “B”) was used for quick isolation of total RNAs. The manufacture’s protocol was followed. Northern Blot 1. Add 5.2 g of agarose in a 500ml flask add 80 ml of 5X MOPS buffer (pH 7) and 250 ml of nanopure water. Put in the microwave and allow to boil throughly to melt all of the agarose. Meanwhile tape the edges of the 20 x 30 cm gel box and put it in the hood and fix the 0.7 cm x 2 mm gel comb in place. When the agarose is melted cool to about 60°C and add 69 ml of 37% formaldehyde while swirling to evenly mix the formaldehyde into the agarose. Pour the mixture in the gel box and smooth out any bubbles. It hardens in about 30 minutes. The concentration of agarose is 1.3% and the gel is 186 about 0.7 cm thick. Up to about 60 pl of sample can be loaded in the slots. 2. While the gel is cooling denature the RNA samples. Normally about 10 pg of total RNA is run but 50 pg will be successfully separate by this method. Prepare the following in a microfuge tube: 5X MOPS buffer 4 pl formaldehyde (37%) 3.5 pl formamide 10 pl RNA 2.5 pl to 4 pl Mix and place at 55°C for 15 min. Add 2 pl of sterile loading buffer (50% glycerol/ 0.05% bromophenol blue). Make sure that the RNA is assayed correctly for concentration or else the lanes may have different amounts. It is best to determine the OD of all then make a dilution to 1 or 2 mg/ ml for all samples. Reassay the OD of the dilution then calculate the amount load. 3. Gently remove the comb from agarose gel. Fill up the Chambers in the gel box which require about 800 ml per side and add a covering layer of buffer to about 1 mm to just cover the gel and fill up the slots. Do not overfill because the formaldehyde concentration must remain high. Add the samples and run the gel at about 40 volts overnigh (ca. 11.5 hrs with the EC 500; use 50 V for the EC 600) so that the blue dye has run about 10 cm. Run all of the gels the same way so that in the future you can compare gels run on separate days. Optional faster runs: 80 V 5.5 h 10 cm EC 500 187 100 V 5.5 h hrs 10 cm EC 600 Save the running buffer for reuse: can be used at least 3 times. 4. Cut out the gel area containing the lanes and put it in another box and rinse with 1 liter of the Tris or phosphate solution.. 5. Meanwhile turn on the Haake Circulator cooling bath set at -10°C. 6. Replace with 1 L of the Tris or phosphate buffer solution and shake for 15 min. and then pour that out and replace with one more liter of Tris or phosphate buffer. Shake again for 15 min plus add 5 drops of 5mg/ ml Ethidium bromide to stain the RNA. The rRNA on the gel is usually not visible in the stained gel but it will be stained on the blot. 7. Remove the gel and look at the RNA on the ultraviolet light box. The RNA will only be visible well if the Tris buffer had been used and pH is adjusted to 8. The ribosomal RNAs of 26S and 175 should be visible about 5 to 7 cm from the slot. Trim away any extra gel outside of the lanes and cut off the gel below the bromphenol blue area at the bottom of the gel. Good visualization at this atage is not really necessary. The bands will appear well on the membrane after blotting. 8. Cut out 4 pieces of Whatrnan 3MM paper and one piece of Gene Screen or Gene Screen Plus the same size as the gel and soak these in the Tris (Gene Screen Plus) or phosphate (Gene Screen) buffer. Assemble the electoblot sandwhich in a large plastic box. First add about 1.5 liter of the appropriate buffer and soak one of the sponges. Then add 2 sheets of the paper making sure that no air bubbles are trapped between or under them. Add the gel on top of 188 them while everything is submerged in the buffer to prevent trapping of air bubbles and then put the Gene Screen or Gene Screen Plus on tap of gel. Smooth out the layers and then add the final two sheets of paper under the buffer as before. Add the other side of the sandwich and place in the electroblot aparatus which is filled with 5 L of the buffer. 9. The Gene Screen side of the sandwich is on the positive side of the electoblotter. Start the electrobloting at 0.4 amp for one hour and Check the amperage in about 15 min to make sure that it is stable and still on. After one hour increase the amperage to ca. 0.9 amp for 3 hours. Remove the blot and air dry or briefly dry on the filter paper and then bake at 80°C for 2 hours. The air drying step is a good overnight stopping point. Look at the blot under the short wave length uv light and mark the slots, the rRNA bands, the date and the samples in pencil on the blot. 10. Prepare the prehybridization buffer in a 50 ml tube. 50% formamide 25 ml 50X Denhardt’s 501 5 ml Do not heat over 37° to thaw. 1 M Tris, pH 6.8 2.5 ml NaCl 2.92g Dissolve NaCl first in the aqueous ingredients not the formamide or SDS. 10% SDS 5 ml 50% dextran sulfate 10 ml 10 mg/ml salmon DNA 0.5 ml 189 Check pH with paper. Should be 7 and pale green. If not and the pH is very green the formamide may need to be deionized and the buffer should not be used for hybridizition. 50% dextran sulfate = 100 g plus 155 ml of nd water; heat and stir to dissolve slowly. 11. Seal the blot in the seal a meal bags and add 10 ml of the prehybridization buffer. Place in a 42°C water bath and allow the bubbles to float up to the top. Prehybridize at least one half hour but can be 0/ n or longer. 12. Add the probe to 10 ml of the prehybridization buffer so that the probe concentration is s 10 ng/ ml with a specific activity of at least 1 x 107 per pg. Usually add one half of the oligolabelling reaction of 100 ng DNA. Heat the hybridization buffer for about 10 min in boiling water to denature the DNA then quickly cool on ice briefly. Pour off the old prehybridization buffer and add the cooled hybridization buffer to the blot and seal again. Leave in the 42°C bath for two days before the posthybridization washes. Turn on the 65°C shaker bath before the posthybridization so that it heats up in time. 13. Remove the hybridization buffer andsave in a plastic tube at room temperature (can be reused at least 3 times within 2 weeks). Wash the blot in 1 L of 2 X SSC twice for 5 min with shaking at RT. Follow with a wash in 1 1 of 0.5 X SSC/ 1 % SDS (Sigma 95% SDS) at 65°C for 30 min with shaking. Check counts of blot with Geiger counter held over the top of blot for background which should be low and over the lower part below the rRNA band which should be 190 noticeabley higher. Usually necessary to wash again at 0.5 X SSC/1 % SDS at 65°C for another 30 min. Remove excess SDS by several quick washes over 30 min with 0.1 X SSC at RT. During the washes do not allow the blot to dry. 14. Wrap the blot in plastic wrap and then mount on 3MM paper. Do not get the outside wet or it will stick to the film during exposure. Mark the rRNA bands location, the date and the orentation of the lanes and someother X marks on the paper with ink spiked with some 3SS. Place into light proof cassette against XARS film with the film against an intensifying screen. Expose at -70° for at least 1 day. Sequencing ( Sanger, et al., 1977 ) Double stranded DNA sequencing is time and labor saving comparing to ss-DNA sequencing. By using the following protocol, we can easily read up to 300 bp. 1. PREPARATION OF DOUBLE-STRANDED DNA FOR SEQUENCING See Plasmid mini-prep and large preparation DNAs which were isolated by mini and large prep except quick mini prep could be used for sequencing. II. DENATURATION OF PLASMID FOR SEQUENCE ANALYSIS 1. Take 10 pg. ds plasmid in TE buffer in 1.5 ml microfuge-tube or use all of one rnini-prep plasmid DNA. 191 2. Add up to 0.4 M NaOH, then incubate at 37°C for 15 min. 3. Add 0.1 vol of 3 M NaAC (pH 5.0) to neutralize. 4. Add 2 to 4 vol 100% EtOH (cold), then precipitate. at -70°C for 30 to 60 min or overnight. 5. centrifuge for 15 min at 4°C at 13,000 rpm. 6. Wash with 300 pl of 70% EtOH for 3 min, then spin 3-5 min at micro-centrifuge. Pour out EtOH and remove residual EtOH with micro-pipette. 7. Dry the pellet at room temp. for 30 min. 8. Keep the sample dry at 4°C until ready for sequencing. 9. Before the sequencing, add 7 111 dde to dissolve the DNA. 10. Add 2 ul 5X sequence kit buffer (United State Biochemical), 1 pl Primer (about 40 ng) and mix with micro-pipette. 11. Put the tube in 65°C water bath for 2 min., then cool at room temp to < 30°C. (about 30 min) 12. Follow the USB brief protocol. 13. We follow the Sequenase Version II protocol supplies by USB modofied as follows: 1) use 1:2 dilution of labelling mix (instead 2:8) 2) the termination reaction is done between 40-45°C (instead of 37°C) for 5 min. 111. Gel PREPARATION and ELECTOPHORESIS 1. Thoroughly mix the following components: 6% : 50.4 g. Urea 6.84 g. acrylamide (Ultrapure) 0.36 g. bisacrylamide (Ultrapure) 192 12 ml 10X TBE 0.12g ammonium persulfate. 68 ml dHZO 120 ml 2. Filter (Whatrnan No.1) the solution into a flask on ice. 3. Place gel solution on room temp. until the temperature of the solution reaches 15°C. 4. Add 60 pl of TEMED and swirl gently to ensure thorough mixing, and pour the gel immediately. 5. Insert the comb at the top of the gel in a horizontal position and Clamp the plates at the top. Leave the gel flat on the bench horizontally for about 30 min. 6. Buffer preparation: 1 X TBE 1.6 L. 7. After the gel is polymerized, tear off the tape of the bottom and affix plates to the sequencing appratus. Pour running buffer (1X TBE) into upper and lower reservoirs. 8. Remove one comb and flush wells with I X buffer to remove any unpolyrnerized acrylamide; insert one shark-tooth comb in the same position; then, do the same for the other comb. 9. Pre-run the gel for I to 2 hours at 60 Watt (constant power). (The running power is 50-55 Watt.) The optimal plate temerature (measured with a thermometer outside the plate) for a good running is about 40°C. 10 Check the leakage of the wells by loading 1 111 stop buffer. (prepare samples while pre-run. See brief protocol supplies by manufacture) 193 1 1. After electrophonesis is finished, fixing 15 min. (15% methanol/ 5% acetic acid) with gentle shaking every 5 min. and drying at 80°C for 40—50 min. 12. Place against XARS film and expose overnight. References Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979). Biochemistry 18, 5294-5299. Feinberg, A. P. and Vogelstein, B. (1983). Anal. Biochem. 132, 6-13. Sambrook, J ., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY. Sanger, F., Niklen, S., and Coulson, A. R. (1977). Proc. Nat. Acad. Sci. USA 86, 4076-4080.