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The developmental expression of the Dictyostelium discoideum ras gene and preliminary detection of a… Gray, Virginia Elaine 1987

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DEVELOPMENTAL EXPRESSION OF THE DICTYOSTEL IUM DISCOIDEUM RAS GENE, AND PRELIMINARY DETECTION OF A SECOND RAS-HOMOLOGOUS B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES Department of M i c r o b i o l o g y We accept t h i s t h e s i s as conforming to the required' standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1987 ( c ) v i r g i n i a E l a i n e Gray, 1987 SEQUENCE IN ITS GENOME. - By VIRGINIA ELAINE GRAY MASTER OF SCIENCE l n In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6G/81) ABSTRACT The expression of a mammalian ras gene analog was previously found by Reymond et a l . to be developmentally regulated in Dictyostelium  discoideum using Northern analysis of strain AX-3 RNA (1984, Cell 39;141) and by Pawson et a l . using specific immunoprecipitation of in vivo synthesized proteins from strain V12M2 (1985, Mol. Cell Biol. 5;33). Due to differences in the results of the two studies, i t was decided to further examine ras expression by applying both protein and RNA techniques to a single strain of D.discoideum, V12M2. RNA samples from strain V12M2 cel l s at different stages of development were analyzed using Northern blotting. The same RNAs were translated _in vitro, and the ras proteins synthesized were immunoprecipitated and analysed by polyacrylamide gel electrophoresis. In agreement with the findings of Reymond et a l . (1984, Cell 39;141), Northern analysis with the cDNA ras probe revealed that the highest levels of the 1.2 and 0.9 kb ras mRNAs were present in the total RNA of V12M2 cel l s at the pseudoplasmodial stage of development, and very l i t t l e ras mRNA was present in early developing c e l l s . In contrast to the Northern analysis the greatest amount of ras protein was rn vitro translated from the RNA of vegetative and 2 hour ce l l s . Hence this work confirms in a single strain of Dictyostelium that the greatest amount of ras protein is synthesized at those developmental stages that contained the lowest levels of mRNA detectable by the cDNA probe. Possible reasons for this phenomena are discussed. i i In vitro RNA translation was also used to study the relationship between the two ras proteins of 23 and 24 kd. The proteins did not appear to be derived from one another by degradation or by post-translational modification. This result suggested that the two ras proteins of strain V12M2 must be derived from two different mRNAs. High stringency Southern blots of AX-3 DNA showed the expected restriction fragments detected by Reymond et a l . (1984, Cell 39_;141) . Low stringency blots showed three faint additional restriction fragments in Eco RI digests of AX-3 DNA. No additional restriction fragments were generated by an Eco RI-Bgl II digest, but two of the three faint bands were smaller. This suggested that at least two of the Eco RI ras fragments are non-contiguous, and hence two to three ras genes may be present in addition to the one characterized by Reymond et a l . (1984, Cell 39;141). A l l Northern and Southern bolts were probed with antisense RNA probes in order to gain greater sensitivity of detection as described by Cox et a l . (1984, Dev. Biol. 101;485). i i i TABLE OF CONTENTS Abstract i i Table of Contents i v L i s t of Figures v L i s t of Abbreviations v i Acknowledgements Introduction • 1 Materials and Methods 18 Results 18 Discussion 49 Bibliography 55 i v LIST OF FIGURES Figure l a . Northern blot showing the developmental expression of D.discoideum ras in whole RNA from strain V12M2, probed with Dd-rascl RNA 29 Figure lb. Northern blot with resolution of the two D.discoideum ras RNAs 29 Figure 2. Southern blot of AX-3 DNA, probed at high stringency with Dd-rascl 32 Figure 3. Southern blot of AX-3 DNA, probed at low stringency with Dd-rascl 33 Figure 4. ras proteins immunoprecipitated from iri vitro translations of developmental time-point RNA, from D.discoideum strain V12M2 37 Figure 5. A comparison of the rates of migration of ras proteins synthesized _in vitro and in_ vivo 38 Figure 6. Effect of protease inhibitors on ras proteins translated _in_ vitro 40 Figure 7. Ratio of ras proteins seen when vegetative RNA is translated _in_ vitro for increasing lengths of time 42 Figure 8. Ratio of ras proteins seen when 2 hour RNA is translated rn vitro for increasing lengths of time 43 Figure 9. Ratio of ras proteins seen when 12 hour RNA is translated _in vitro for increasing lengths of time 44 Figure 10. Ratio of ras proteins seen when 15 hour RNA is translated _in vitro for increasing lengths of time 45 Figure 11. Plot of amounts of ras proteins immunoprecipitated from vn vitro translation reactions with developmental time-point RNAs 47 v LIST OF ABBREVIATIONS cAMP cyclic adenosine 3',5' monophosphate c-Ha-ras cellular homolog of the v i r a l Harvey ras gene c-Ki-ras cellular homolog of the viral Kirsten ras gene DAG 1,2-diacylglycerol Dd-rascl cDNA clone of Dictyostelium ras RNA isolated by Reymond et a l . (1984, Cell 39, l^l) EGF Epidermal Growth Factor GDP guanosine diphosphate GTP guanosine triphosphate GTPase guanosine triphosphatase HMSV Harvey Murine Sarcoma Virus IPj inositol-l,4,5-phosphate kd kilodaltons LTR Long terminal repeat (of retrovirus genome) mRNA messenger RNA MLV Murine Leukemia Virus MSV Murine Sarcoma Virus MTV Murine Tumor Virus NRK Normal Rat Kidney PIp2 phosphat i dyli nos i tol-4,5-biphosphate poly A+ RNA polyadenylated RNA RaRIg rabbit anti rat immunoglobulin G rRNA ribosomal RNA S phase DNA synthesis phase of c e l l cycle SSV Simian Sarcoma Virus tRNA transfer RNA vi I wish to express my g r a t i t u d e to my s u p e r v i s o r s Dr. Tony Pawson and Dr. Gerry Weeks f o r p r o v i d i n g me with the o p p o r t u n i t y to do t h i s work. I a l s o wish to thank my parents and Ralph McLean f o r t h e i r s u p p o r t . v i i INTRODUCTION The conservation of oncogenes in the genomes of species ranging from vertebrates to the simplest eucaryotes suggested they played an important cellular role (Hunter, 1984). The transforming and tumorigenic effects of the v i r a l oncogenes, and the latent potential of their cellular protooncogene homologs for "activation" to oncogenic forms suggested these genes controlled c e l l growth. Indeed, several oncogenes have now been recognized as analogs of growth factors (Waterfield et a l . , 1983; Doolittle et a l . , 1983), growth factor receptors (Downward et a l . , 1984; Sherr et a l . , 1985) or belonging to a family of tyrosine kinases similar to the EGF receptor (Hunter, 1984) . The ras members of the oncogene family were discovered as sequences carried by the Harvey and Kirsten murine sarcoma viruses that were responsible for tumorigenesis (El l i s et a l . 1980, 1981): The ras gene has been of particular interest in cancer research as i t s activation may be responsible for 10 to 30% of a l l human malignancies (Lacal and Aaronson, 1986a; Finkel et a l . , 1984). An analog of the mammalian ras gene was found in the genome of the cellular slime mold Dictyostelium  discoideum (Reymond et a l . , 1984). This organism has a simple developmental program that causes a population of undifferentiated single-celled amoebae to aggregate upon depletion of nutrients, and form a multicellular organism with two differentiated c e l l types. The cells undergo two periods of mitosis and DNA synthesis during development (Zada-Hames and Ashworth, 1978). Since there is some evidence to suggest 1 that ras function is important in the ini t i a t i o n of the S phase of the ce l l cycle (Feramisco et a l . , 1984; Mulcahy et a l . , 1985), and since in Dictyostelium there is some evidence that c e l l fate may be linked to the ce l l cycle (Weijer et a l . , 1984a; Sharpe et a l . , 1984) this organism is an interesting yet simple model in which to investigate a possible linkage of ras expression to the c e l l cycle as well as to differentiation. Indeed, ras has been already been found to be differentially expressed in D.discoideum, both temporally, and with respect to the two c e l l types (Pawson et a l . , 1985; Reymond et a l . , 1984). D.discoideum is amenable to transformation techniques (Barclay et a l . , 1983; Reymond et a l . , 1985) but does not have the advantage of si t e -directed transformation by gene replacement, as in yeast (Rothstein, 1983) . The v i r a l and mammalian ras genes encode 21 kd proteins (p21 ras) , known to bind GTP and GDP with high af f i n i t y (Shih et a l . , 1980), and to have low GTPase activity (McGrath et a l , 1984; Sweet et a l . , 1984). Both the Harvey and Kirsten murine sarcoma virus (MSV) ras proteins have threonine in place of the normal alanine at position 59, and autophosphorylate at that site (Papageorge et a l . , 1982). The ras protein undergoes several post-translational changes. Harvey MSV p21 ras is derived from a larger precursor (Shih et a l . , 1982). Palmitic acid i s covalently attached (Buss and Sefton, 1986) near the carboxyl terminus, most likely to cysteines at positions 184 or 185 (Willumsen et a l . , 1984) and is required to bind the ras protein to the inner surface of the plasma membrane. Transformation by activated forms of p21 ras w i l l occur 2 only i f the protein is in association with the plasma membrane. Proteins lacking amino acids 184 to 189 have no l i p i d attached, remain in the cytoplasm, and f a i l to transform NIH-3T3 cells (Willumsen et a l . , 1984). Since a portion of Harvey MSV p21 protein is autophosphorylated, and a portion of each of those species is fatty acylated, Buss et a l . (1986) were able to resolve four distinct forms of HMSV ras protein in addition to the precursor. The ras protein is striking in that the normal cellular form of the protein can be "activated" to i t s oncogenic form by single point mutations at position 12 (Tabin et a l . , 1982; Reddy et a l . , 1982; Capon et a l , 1983; Shimizu et a l . , 1983) that cause a reduction in GTPase activity (McGrath et a l . , 1984), or at position 61 (Yuasa et a l . , 1983) that is likely part of the GTP binding site. There are 3 amino acid differences between the v i r a l and cellular Harvey ras proteins, and 7 between the viral and cellular Kirsten ras proteins (Shimizu et a l . , 1983), that in each virus include a position 12 mutation and the substitution of threonine for the native alanine at position 59. Either change alone is sufficient to cause oncogenic activation (Lacal et a l . , 1986b). The effect of threonine at position 59 appears to be negated by the _in vitro mutagenesis of asparagine 116 to either lysine or tyrosine, as this second mutation abolished guanine nucleotide binding, autophosphorylation, and transformation of cells (Clanton et a l . , 1986). Additional single amino acid changes sufficient to cause oncogenic activation were identified at positions 13 and 63 by mutagenesis (Fasano et a l . , 1984). 3 The ras sequences are so conserved between species that ras proteins from mammalian cells w i l l function in yeast c e l l s (Kataoka et a l . , 1985). Transfection of growing mouse NIH-3T3 or normal rat kidney (NRK) cells with an activated human Harvey ras gene produces foci of transformed cel l s , whereas the normal human Harvey ras gene produces l i t t l e or no effect. However, both the normal murine and the normal human Harvey ras genes could transform NIH-3T3 mouse cells i f transcribed in very high levels from a highly active viral LTR promoter (DeFeo et a l . , 1981, Chang et a l . , 1982) . Unexpectedly, quiescent mouse NIH 3T3 c e l l s , quiescent normal rat kidney c e l l s and rat embryo fibroblasts (REF-52) a l l underwent morphological changes and initiated DNA synthesis in depleted media, after the microinjection of the oncogenic form of the human Harvey ras protein (Feramisco et a l . , 1984). Feramisco et a l . found that microinjection of the same levels (10^ to 10^ molecules per cell) of the normal human Harvey ras protein had l i t t l e effect. However, Stacey and Kung (1984) reported some foci showing less pronounced transformation upon microinjection of high levels (J=6X10^  molecules per cell) of the normal human Harvey ras protein. This result was consistent with the view of Defeo et a l . (1981) that elevated levels of the normal ras protein could lead to transformation. These findings suggest that the ras protein provides a signal to cells to enter the S phase of the c e l l cycle. Transformation was inhibited by both cycloheximide and actinomycin D, indicating a requirement for protein synthesis and transcription to mediate the transforming effects of ras (Feramisco et 4 a l . , 1984) . It should be noted that not a l l c e l l lines respond to p21 ras injection (Feramisco et a l . , 1984; Stacey and Kung, 1984) or transfection by ras DNA (Sager et a l . , 1983). Cell lines such as NIH-3T3, which are highly amenable to transformation by ras via transfection or microinjection, may be in a penultimate stage of transformation or have cell-type specific properties that make them susceptible to ras stimulation (Land et a l . , 1983). As an-extension of prior experimentation with microinjection of ras proteins, Mulcahy et a l . (1985) microinjected the anti-ras monoclonal antibody Y13-259 into quiescent NIH-3T3 cells prior to the addition of 10% fetal calf serum, and found that the antibody (used at about 3x10° molecules/cell, the equivalent of 0.2% of c e l l protein) abolished the DNA synthesis which would otherwise occur about 16 hours after the addition of fresh serum. DNA synthesis was not prevented by microinjection of either an anti-human interferon antibody or the monoclonal antibody Y13-238. This latter antibody is known to bind Harvey ras but not Kirsten ras proteins. That Y13-238 serves as a negative control' in this experiment suggests that the endogenous cellular Kirsten ras (c-Ki-ras) protein was able to function in place of the c-Ha-ras protein, whereas binding of both c-Ha-ras and c-Ki-ras proteins by Y13-239 prevents transduction of a signal for DNA synthesis. This work adds to the evidence that the ras protein is required by cells to enter the S phase of the c e l l cycle. Recently, i t was demonstrated that pre-binding of Y13-259 antibody to 5 bacterially produced Ha-ras and Ki-ras proteins did not affect GTP binding, GTPase activity, or autophosphorylation (Lacal and Aaronson, 1986b). This implied that since Y13-259 binding does not interfere with these biological functions, i t may neutralize ras function _in_ vivo by binding to a site on the protein otherwise used to interact with another factor involved in signal transduction. Lacal and Aaronson (1986b) also used 3' deletions to localize the Y13-259 binding s i t e to ras amino acids 69-89. Using this information, Papageorge et a l . (1986) made mutant ras proteins lacking amino acids 64-72, 69-72, 72-76, and 72 alone, that escaped immunoprecipitation by Y13-259 and yet retained transforming a b i l i t y . It was thought that methionine-72 was the c r i t i c a l component in the Y13-259 epitope. If the interaction site proposed by Lacal and Aaronson (1986b) exists, i t can not require the region from amino acids 64 to 76. Recent work by Papageorge et a l . (1986) showed that substitution of the normal position 12 glycine had no effect on the GTP binding associated with the region around 59-61, and the substitution of threonine for alanine at 59 had no effect on the GTPase activity associated with position 12. Transfection of NIH-3T3 c e l l s with ras DNA encoding either glycine 12-threonine 59 or lysine 12-alanine 59 produced foci with equal efficiency, demonstrating that either position 59 changes alone, or lower GTPase activity alone were sufficient to cause transformation. The threonine 59 mutation also increased GTP binding by 3 fold, although this increased binding did not affect GTPase activity in the assay performed. 6 Similarities between the ras proteins and the G proteins (the regulatory subunits of adenylate cyclase) led to a hypothesis that ras similarly acted on adenylate cyclase, to control intracellular cAMP levels (Sefton et a l . , 1982). The 45kd alpha stimulatory (G*s) and alpha inhibitory (G<*i) G proteins reside in the membrane and have sites for ADP-ribosylation and GTP binding. When a stimulatory hormone binds i t s receptor, the receptor binds the G*s subunit, increasing the aff i n i t y of the latter for GTP. The entire complex binds adenylate cyclase which synthesizes cAMP until the hydrolysis of GTP by the G<*s subunit terminates the stimulatory effect. The importance of this last step is demonstrated by the irreversible stimulation of the enzyme in the presence of non-hydrolysable GTP analogs (Gilman, 1984) . Both the G and ras families of proteins are localized on the inner surface of the plasma membrane, both bind guanine nucleotides with high a f f i n i t y (Scolnick et a l . , 1979), and both have weak GTPase activity (McGrath et a l . , 1984; Sweet et a l . , 1984). The ras proteins also have a small degree of amino acid sequence homology with the bovine Gi subunit (Hurley et a l . , 1984). The discovery that position 12 activated forms of the ras protein had a 7 or 8 fold reduced GTPase activity relative to wild type (McGrath et a l . , 1984, and Sweet et a l . , 1984) led to an attractive model of oncogenesis, wherein ras activation would result in elevated levels of cAMP. This would overstimulate cAMP-dependant protein kinase, thought to be the only mediator of cAMP signals in eucaryotic c e l l s . One of the regulatory subunits of cAMP-dependent protein kinase, RII, was found to possess topoisomerase I activity (Constantinou et a l . , 1985), which in 7 turn appeared to induce transcriptional activity in Drosophila polytene chromosomes (Fleishman et a l . , 1984), suggesting one way in which cAMP signals may be transduced to the nucleus. This model appeared to be born out by the work of Toda et a l . (1985) with Saccaromyces cerevisiae, an organism possessing two ras gene homologs. The levels of cAMP synthesized in the yeast reflected the number of functional ras genes, such that yeast lacking one ras gene, especially RAS2 showed low levels of cAMP, yeast with two ras genes showed normal levels of cAMP, and yeast that had their normal RAS2 gene replaced with a valine 19 activated ras gene (equivalent to the v i r a l valine 12 mutation) showed highly elevated levels of cAMP. Yeast lacking both RASl and RAS2 were non-viable unless rescued by the bcyl ("bypass cAMP") mutation which results in a cAMP-independent protein kinase. These "rasl~ras2~bcyl" c e l l s had low levels of cAMP, but were viable. Finally, yeast with only one RAS valine 19 gene were viable, but were unable to prepare properly for sporulation and failed to accumulate the carbohydrates associated with this process. This implied that cAMP levels must decline during the signalling to end vegetative growth and prepare for sporulation. The requirement of ras genes for cAMP production and v i a b i l i t y in yeast is not universal. Low stringency hybridizations of DNA from another species of yeast, Schizosaccharomyces pombe, indicated the existence of only one ras gene. When this site was disrupted by replacing the rasl gene with selectable leul or ura4 markers, these yeast produced equal numbers of ra s l - and rasl+ spores, a l l of which were 8 viable. Furthermore, levels of intracellular cAMP detected in both r a s l -and rasl+ yeast were normal. But r a s l - haploid yeast could not mate, and rasl-/rasl- diploids could not sporulate effi c i e n t l y (Fukui et a l . , 1986). Unless a second ras gene has gone undetected in this species i t would appear that ras is not universally required for v i a b i l i t y in yeast, although its requirement in spore formation is implicated in both strains. The work of Beckner et a l . (1985) provided evidence that the ras protein was not a regulatory component of adenylate cyclase in mammalian cells , because i t could not "rescue" a CXs- mutation in the human lymphoma line S49, which lacks the stimulating regulatory subunit of adenylate cyclase and requires exogenous cAMP for v i a b i l i t y . cAMP could be synthesized, in vitro u t i l i z i n g adenylate cyclase activity from solubilized S49 membranes, and G«<s activity from normal c e l l membranes. The latter had their endogenous adenylate cyclase activity abolished during an incubation. S49 membranes alone, or S49 membranes with bacterial or v i r a l p21 (shown to retain GTP binding, GTPase activity and autophosphorylating activity _in vitro) could not synthesize cAMP in this assay. There was also speculation that the p21 protein might serve as the G«i subunit, because cAMP levels are often below normal levels in epithelial and fibroblast cells lines transformed with Harvey or Kirsten ras. Yet bacterially-produced ras protein did not decrease the rate of cAMP synthesis in soluablized membrane assays of either normal or Harvey HSV transformed c e l l s . Further convincing evidence that ras does not interact with adenylate 9 cyclase in cells other than yeast, comes from work done with Xenopus oocytes. Oocytes removed from Xenopus ovaries are arrested in the prophase of meiosis, and agents which lower cAMP levels, such as insulin or the physiological inducer progesterone, w i l l induce meiosis. Agents which increase cAMP levels, such as cholera toxin, phosphodiesterase inhibitors, or cAMP-dependent protein kinase inhibit maturation. Birchmeier et a l . (1985) found that microinjected ras protein stimulated maturation, and furthermore, that valine 12 mutants (which would increase cAMP levels according to the model of Toda et a l . , 1985) were 100 fold more potent in bringing about meiosis. This suggested that the valine 12 ras protein must not be causing an increase in cAMP levels, indeed; no increase or decrease was detectable. Hence, in Xenopus, ras doesn't appear to interact with either adenylate cyclase or cAMP. The cellular effects of ras activity outlined above w i l l doubtless be re-examined in light of recent evidence that ras couples a second group of hormones that includes bombesin, bradykinin, and gastrin releasing peptide (GRP) to phospholipase C (Fleischman et a l . , 1986). This class of hormones act not via adenylate cyclase and cAMP, but via phospholipase C, which hydrolyzes phosphatidylinositol-4,5-biphosphate (PIP 2), a l i p i d component of the c e l l membrane into two breakdown products, both of which appear to have regulatory activity. Inositol-1,4,5-triphosphate (IP^) causes the release of Ca^+ into the cytosol from intracellular stores (Berridge and Irvine, 1984; Streb et a l . , 1983). 1-,2-diacylglerol (DAG) activates protein kinase C (Bell et a l . , 1979; Nishizuka, 1984). Both Ca^- release and protein kinase C activate a plasma membrane Na+-H+ 10 exchanger (Burns and Rosengurt, 1983; Moolenaar et a l . , 1984). This results in an elevation of cytoplasmic pH and Na+ levels, c r i t i c a l events in the growth factor stimulation of c e l l proliferation (Habenicht et a l . , 1981; Berridge et a l . , 1984). GTP is essential for the phospholipase C activity required to precipitate these events (Cockcroft and Gomperts, 1985). Fleishman et a l . (1986) found that the levels of the breakdown product DAG to its precursor PIP2were 2.5 to 3-fold higher in NIH 3T3 and NRK cells transformed with v i r a l and position 12 activated ras genes. In a more elegant experiment, Wakelam et a l . (1986) transformed NIH 3T3 cells with the normal human N-ras gene under the control of the dexamethasone inducible MMTV promoter. Dramatic increases in IP3 levels were seen in cells treated with both dexamethasone and those growth factors which act via phospholipase C activation, in particular bombesin, and gastrin releasing protein and bradykinin. Neither dexamethasone nor growth factors alone produced notable increases I P 3 pools, showing a requirement for coupling between ras and the growth factors involved, in order for PIP 2 to be hydrolyzed to IP3. Hence, ras may serve as the intermediary between a class of growth factors and phospholipase C, to allow c e l l s to enter the S phase of the ce l l cycle. Activating mutations of the ras gene may encode ras proteins that act on phospholipase C independent of stimulation by growth factors. The properties of a given c e l l line may also influence the effects of ras gene activation. Cells of the human breast cancer c e l l line MCF-7, for example, produce tumors in nude mice and in ovarectomized nude mice given estrogen, showing a requirement for estrogen for tumorigenicity. 11 Transfection of MCF-7 cel l s with the activated v i r a l Harvey ras gene produces cells that form tumors in ovarectomized mice in the absence of estrogen (Kasid et a l . , 1985). This effect contrasts with the ras-stimulated initiation of mitosis in quiescent NIH 3T3 fibroblasts, and of meiosis in Xenopus oocytes. In a l l three examples, a normal requirement for growth factors can be abolished by a ras gene expressed in elevated levels, or in an activated form. Dictyostelium is an interesting model in which to study the connection between ras expression and c e l l division because mitosis occurs in two distinct periods during development (Zada-Haraes and Ashworth, 1978), and because c e l l fate may be linked to the c e l l cycle (Feramisco et a l . , 1984; Mulcahy et a l . , 1985). The f i r s t period of mitosis peaks at 4 hours of development (beginning at 0 hours and lasting until the f i r s t signs of rippling at 8-9 hours), the second period of mitosis starts with a pronounced peak at f i r s t finger stage which declines towards culmination (Zada-Hames and Ashworth, 1978). D. discoideum has been of interest to biologists primarily for i t s a b i l i t y to exist either as a population of single c e l l s , or as a multicellular organism displaying two differentiated c e l l types. Within hours after the food supply has been depleted, amoebae aggregate to form mounds containing about 10 c e l l s each. By 14 hours of development the aggregated c e l l s form a migrating slug, with cells which are partially differentiated into the stalk c e l l s of the mature structure (prestalk cells) localized at the anterior of the slug, and the "prespore" c e l l s localized at the posterior end. By 24 hours of development the prespore cells are borne aloft by the upwardly 12 elongating stalk, to form the mature fruiting body. Cyclic AMP in D.discoideum functions in chemotaxis, in the transcription of post-aggregation dependant mRNA species (Chung et a l . , 1981), and in the maintenance of expression of many developmental genes (Barklis and Lodish, 1983) . About 6 hours after the depletion of nutrients, amoebae begin to stream towards individual c e l l s in the population which emit periodic pulses of cAMP. The cells produce an extracellular and a c e l l surface phosphodiesterase which break down extracellular cAMP. The balance of these factors creates pulsatile signals and a chemotactic gradient, emanating from the center of each aggregation territory. The expression on the c e l l surface of both cAMP binding protein and cAMP phosphodiesterase remain low until 6 hours of development, rise to sharp peaks when aggregation is complete at 8 hours, and then decline towards culmination (Henderson, 1975) . This reflects the requirement of the cAMP signalling system to establish aggregates. The secretion of cAMP i t s e l f rises steadily from 9 hours, peaks at 15 hours, and then declines sharply (Tyler and Bonner, 1969). This may reflect a lower requirement for cAMP to maintain the differentiated state of prestalk and prespore cells as they approach terminal differentiation. In rapidly shaking cultures, where c e l l - c e l l adhesion is inhibited, cAMP alone appears sufficient to induce the transcription of prestalk-specific genes, whereas both cAMP and c e l l - c e l l contact are required to induce the transcription of prespore-specific genes (Mehdy et a l . , 1983). Another factor identified as necessary for ce l l - s p e c i f i c gene activation is DIF (differentiation inducing factor), a dialyzable, l i p i d - l i k e factor 13 which seems to be required for the formation of stalk cells (Kopachik et a l . , 1983). The disaggregation of partially developed c e l l masses causes a rapid loss of cell-type specific gene transcription, and a reduction of some aspects of gross c e l l morphology such as prestalk and prespore vacuoles. When disaggregated ce l l s are prevented from reaggregating in fast shake-suspension cultures, the addition of exogenous cAMP restores partially or completely the transcription of a l l cell-type specific genes. Recovery of expression of cell-type specific RNAs was not seen in cultures that did not receive exogenous cAMP (Barklis et a l . , 1983). Chung et a l . (1981) derived a h a l f - l i f e of 4 hours for both vegetative and aggregation-dependant mRNA in pseudoplasmodial stage c e l l s . When pseudoplasmodial stage cells were disaggregated, the h a l f - l i f e of only the aggregation-dependant mRNAs was reduced to 25 to 45 minutes. Possibly, RNase activity is important in ending the expression of aggregation-dependant mRNA in disaggregated c e l l s . Many differences are apparent in the role of cAMP in Saccharomyces  cerevisiae and in D.discoideum. In S.cerevisiae, low cAMP levels appear to signal both the depletion of nutrients and the preparation for sporulation. The expression of a position 12 activated ras gene in S.cerevisiae appeared to interfere with the preparation for sporulation by causing constituitively high levels of cAMP (Noda et a l . , 1985). Conversely, in D.discoideum the cellular response to depletion of nutrients is a prolonged period of elevated cAMP synthesis. Furthermore, elevated cAMP levels are required to initi a t e and maintain the 14 differentiated state of prespore c e l l s . Hence, i f ras acts on adenylate cyclase in Dictyostelium, i t does so in a manner vastly different to that in S.cerevisiae. A Dictyostelium discoideum ras gene originally isolated by Reymond et a l . (1984) as a prestalk-specific cDNA, was found to have 64 to 66% protein homology with the ras genes of other species. A probe made from the D.discoideum ras cDNA hybridized to 0.9 and 1.2 kb transcripts in strain AX-3 RNA. Both mRNAs were strongly expressed in the poly A+ RNA of AX-3 cells at 15 and 17.5 hours of development. Levels of the two transcripts declined steadily to zero in the RNA of cells at 20, 22.5 and 25 hours of development. Prior to 15 hours of development, the only detection of a ras mRNA was in RNA from vegetative c e l l s , and only the 1.2 kb transcript was present. A part of the D.discoideum ras gene, encoding amino acids 59 to 187, was cloned into a g t l l expression vector, and the resultant E.coli -galactosidase/ras fusion protein was used to produce a polyclonal anti-ras serum in rabbits. When Western blots of protein from D.discoideum cells at various stages of development were probed with this immune serum, the greatest amount of ras protein appeared in vegetative c e l l s , and levels declined steadily to zero at 25 hours. This pattern of expression was in striking contrast to that seen in Northern blots, and Reymond et a l . (1984) suggested that the ras mRNA may be more labile than the protein. In contrast, Pawson et a l . (1985) analyzed the expression of H I vivo pulse-labelled ras proteins in strain V12M2. The monoclonal antibody Y13-259 immunoprecipitated two Dictyostelium ras proteins of 23 and 24 15 kd. The 24 kd protein was a minor species not always detected, but was expressed predominantly in pseudoplasmodial stage c e l l s when seen. High total levels of ras protein were detected in vegetative c e l l s , followed by a marked increase in synthesis in the f i r s t one to two hours of development. A second smaller burst of expression occurred during pseudoplasmodial formation (at 12 hours of development), after which ras protein levels diminished towards culmination. The 23 and 24 kd Dictyostelium ras proteins appeared closely related to each other as well as to the Harvey v i r a l ras protein, as determined by tryptic peptide analysis. Both the Northern blots of Reymond et a l . (1984) (using strain AX-3) and the immunoprecipitations of Pawson et a l . (1985) (using strain V12M2) showed two peaks of ras expression, one at 0 to 2 hours, and the other at the pseudoplasmodial stage. The most obvious difference between the results of the two labs was that more ras protein was immunoprecipitated in strain V12M2 vegetative cells relative to pseudoplasmodial c e l l s (Pawson et a l . , 1985) whereas far more mRNA was detected in strain AX-3 peudoplasmodial cells relative to vegetative c e l l s . Possible explanations for the lack of correlation between the levels of ras protein and ras mRNA are f i r s t l y , strain differences; secondly, dramatic changes in the efficiency of translation of ras mRNA throughout development; and thirdly, the presence of a second ras gene not detected in the Northern blots of Reymond et a l . (1984), which is actively transcribed in vegetative and early developing c e l l s . In support of this last theory is the finding of Weeks and Pawson (1986) that the ras 16 protein is located predominantly in prespore c e l l s , whereas Reymond et a l . have found the ras mRNA to be predominantly in prestalk c e l l s . It is noteworthy that a l l other organisms thus far studied with the exception of Schizosaccharomyces pombe contain more than one ras gene. It was decided to examine ras expression further by applying both protein and RNA methodologies to a single strain of D.discoideum, V 1 2 M 2 . Levels of ras protein were examined by immunoprecipitating proteins translated in vitro from the RNA used in Northern blots. Low stringency Southern blots were also used to search for the putative second ras gene. Antisense RNA probes were used instead of nick-translated DNA probes, in the hope that the greater sensitivity of the SP6 RNA probe system would allow the detection of heterologous ras mRNA or genomic l o c i . My results show that the highest levels of ras protein are translated in vitro from vegetative and 2 hour RNA, but that the highest levels of ras mRNA are present in the RNA of cells at the pseudoplasmodial stage of development. Hence the level of ras protein synthesized _in vitro did not correspond to .the levels of ras RNA detected at different stages during development. The kinetics of the _in vitro synthesis of the two Dictyostelium ras proteins suggest that they are not derived from one another and hence are translated from two different mRNAs. It is not known whether these two mRNAs are derived from the same ras gene or not. Novel genomic restriction fragments containing ras-homologous sequences were detected by use of an antisense RNA probe under conditions of low stringency. 17 MATERIALS AND METHODS Growth and differentiation of D.discoideum strain V12M2. Amoebae were grown in suspension cultures on E.coli B23 which had been resuspended from M9 media to an optical density at 660 nm of 6 OD in s t e r i l e KK2 buffer (20 mM potassium phosphate monobasic/potassium phosphate dibasic, pH 6.0). Amoebae were generally inoculated at a concentration of 2x10^ cells/ml, and harvested before they had reached a concentration of 6xl0& cells/ml. To induce differentiation, amoebae were separated from bacteria by 3 to 4 differential centrifugations (700g for 2 min), in g KK2 buffer. Amoebae were counted with a haemocytometer, and 10 cells were pipetted onto Millipore f i l t e r s resting on Millipore pads saturated with 0.8 ml KK2 buffer containing 0.5 mg/ml streptomycin. I_n vivo labelling was carried out by transfering f i l t e r s onto a droplet of 250/aCi [35S]-methionine for a two hour period, followed by three washes of the cells in KK2 buffer to remove exogenous label. Extraction of RNA. RNA was extracted using the methods of Blumberg and Lodish (1980). Cells at the appropriate stage of development were washed 8 from f i l t e r s with KK2 buffer and pelleted. Each 2x10 c e l l s was vortexed with 4 ml of lysis buffer containing 50 mM Hepes, 40 mM magnesium -acetate, 20 mM potassium chloride, and 0.2% sodium dodecyl sulfate (SDS). This aqueous phase was immediatedly vortexed with an equal volume of water-saturated phenol. After adjustment to 0.2 M sodium acetate, 4 ml 18 of chloroform was added (per 2x10^ c e l l s ) , and vortexed. The mixture was centrifuged for 10 minutes at 10,000 rpm, and the upper, aqueous layer transfered to a fresh tube. Two more phenol-chlorforra extractions were carried out. The aqueous layer was then extracted twice with two 1.5x volumes of chloroform. Finally, the aqueous layer was brought to 0.3 M sodium acetate, 2 volumes of 95% ethanol was added, and the RNA was precipitated at -20C. Precipitation was often carried out overnight, but 1 hour was sufficient to precipitate milligram quantities of RNA. The RNA was then pelleted 20 minutes at 10,000 rpm, and washed twice with volumes of 75% ethanol equal to the original precipitation volume. The pellet was vacuum-dried, resuspended in s t e r i l e d i s t i l l e d water, and ethanol precipitated a second time. The RNA was pelleted a second time, washed twice in 75% ethanol, vacuum-dried, and resuspended to 2.5 mgs/ml in sterile d i s t i l l e d water. Generally, the RNA yield from early o developing cells was 0.9 to 1 mg per 10 c e l l s , and decreased to 0.5 mg g per 10 cells by 15 hours. Nit r i c acid-cleaned Corex tubes were used throughout. Subcloning ras probes into SP6. Antisense RNA probes were used instead of nick-translated DNA probes, so that the greater sensitivity of RNA probes might allow [in contrast to DNA probes (Reymond et a l . , 1984)] the detection of heterologous ras mRNA and genomic sequences. Cox et a l . (1984) established that the association and disassociation temperatures of RNA duplexes were 20C higher than the corresponding temperatures for 19 DNA duplexes (55C and 75C respectively, for RNA, compared to 35C and 55C for DNA in 50% formamide). Hybridization and washes can be carried out at higher temperatures, and only the antisense strand of the probe becomes labelled by SP6 polymerase, producing a high signal to noise ratio. In formamide concentrations over 40%, RNA:DNA duplexes are also more stable than DNA duplexes, especially as G+C content increases (Casey and Davidson, 1977) . Of interest is the finding of Cox et a l . (1884) that two complementary histone RNAs that differed by 10-15% in their RNA sequences hybridized at 42C, or 13C below the temperature of duplex formation of + and - RNA strands. Constructs: i) The 470 bp PstI fragment of a D.discoideum ras cDNA clone called "Ddrascl", kindly provided by Richard F i r t e l , was excised from pBR322 and ligated into Pstl-digested SP64. Both orientations were recovered. SP6 polymerase transcribes from the antisense orientation, 94 nucleotides of 3' ras gene flanking sequence, followed by the coding sequence corresponding to amino acids 186 to 59. ii) A Harvey murine sarcoma virus (HMSV) ras probe was made by ligating the 700 bp Pstl-Hindlll HMSV ras gene fragment from plasmid "14" (Ell i s et a l . , 1980) into the SP65 vector which had been digested with the same enzymes. SP6 polymerase transcribes approximately 60 nucleotides of v i r a l sequence 3' of the HMSV ras gene, followed by the HMSV ras coding sequence corresponding to amino acids 189 to 1. i i i ) A Kirsten murine sarcoma virus (KMSV) ras probe was made by 20 ligating the 1.2 kb BamHI-Xbal KMSV ras gene fragment from the plasmid "KBE-2" (E l l i s et a l . , 1981) into the SP64 vector linearized with the same enzymes. SP6 polymerase would transcribe the KMSV ras coding sequence corresponding to amino acids 120 to 1, followed by 420 bp of vira l ras 5' flanking sequence. Homology of the three probes to one another (as determined by use of the Delaney SEQNCE program): 1 Harvey MSV ras Kirsten MSV ras Dd-rascl | 53% overall 69% in the f i r s t 100 bp of coding region 61% overall 75% in the f i r s t 100 bp of coding region Kirsten MSV ras | 60% overall 82% in the f i r s t 100 bp of coding region Preparation of radioactively labeled probes. Radioactive probes were prepared as described in the Bio-Can Riboprobe instruction l e a f l e t . 20 jul transcription reactions contained 1.6 ug of linearized template, 12 juM 21 radioactively labelled o6-[32P]-CTP (600 Ci/mmole), lx transcription buffer [200 mM Tris-chloride (pH 7.5), 30 mM magnesium chloride, 10 mM spermidine, and 100 mM sodium chloride], 10 mM DDT, 20 units RNAsin inhibitor, 0.5 mM each of ATP, GTP, and OTP; and 13 units SP6 polymerase (8 units per ug template). Transcription was carried out at 40C for 1 hour. The DNA template was then removed by incubation at 37C for 15 minutes with 0.4 ug DNase and 0.6 p i RNAsin inhibiter. Proteins were removed by one extraction with an equal volume of water-saturated phenol and one extraction with an equal volume of chloroform. The aqueous layer was brought to 2.0 M ammonium acetate, and then 10 ug of carrier tRNA and two volumes of ethanol were added. 4 hours at -80C, or overnight at -20C provided adequate time for preciptation. Probe was pelleted at 10,000 rpm for 45 minutes, and washed once with 70% ethanol. After the probe was dried in a spin-vacuum apparatus and resuspended in 100 ul of s t e r i l e water, 1 ul was removed for Cerenkov counting. The probe was used within a few days of synthesis. Northern blots. 20 ug of total RNA or 3 to 6 ug of poly A+ RNA were dried in Eppendorf tubes and resuspended in 5.3 u l 30 mM sodium phosphate monobasic/sodium phosphate dibasic (pH 7.0), and 8 u l dimethylsulfoxide. 2.7 u l of deionized 40% glyoxal was added to each tube and samples were incubated 1 hour at 50C. 3 ul of loading dye [50% glycerol, 10 mM sodium phosphate monobasic/sodium phosphate dibasic (pH 7.0) , 0.4% bromophenol blue] was added to each RNA immediately prior to 22 electrophoresis. The gel bed and box were soaked in 25 mM iodoacetate for 30-60 minutes, during the preparation of the gel [1.1 or 1.2% agarose in 10 mM sodium phosphate monobasic/sodium phosphate dibasic (pH 7.0), containing 0.5% (w/v) iodoacetate]. The buffer was circulated during electrophoresis with a peristaltic pump. Gels were run at 90 V, after which lanes containing rRNA size markers were excised, soaked 30 minutes in 50 mM sodium hydroxide in order to remove the glyoxal, then soaked 30 minutes in 10 ethidium bromide, and photographed. The gel it s e l f was blotted overnight onto nitrocellulose, which was then baked _in vacuo for 2 hours at 80 C. Northern hybridization was carried out as per Melton et a l . , 1984. Prehybridization was carried out for one to four hours in the following: 50% formamide (deionized overnight on Bio-Rad AG 501-X8 resin and stored at -20C), 50 mM sodium phosphate monobasic/sodium phosphate dibasic (pH 6.5), 5x standard salt citrate, SSC (20x SSC is 3.0 M sodium chloride and 0.3 M sodium citrate, pH 6.8), 0.1% SDS, 1 mM EDTA, 200 ug/ml denatured salmon sperm DNA, and 0.05% Denhardt's reagent [1% Denhardt's reagent is 1% (w/v) each of bovine serum albumin, F i c o l l , and polyvinlypyrolidine]. Hybridization was carried out for a minimum of 16 hours in the same solution, with the addition of 4x10^ cpm of radioactively labeled RNA 2 probe per cm . Three 20 minute washes were carried out in 0.1% SDS, O.lx SSC. Stringent hybridizations were carried out at 55C, and washed at 65C. Low stringengy hybridizations were carried out at 40C, and washed at 40C. 23 Growth of D.discoideum strain AX-3 and extraction of genomic DNA. Cells were grown axenically in shake suspension at 22C in HL-5 media [1.5% w/v Bacto-peptone and yeast extract, 4.3 mM potassium phosphate monobasic/ potassium phosphate dibasic (pH 7.0), 4.3 mM sodium phosphate dibasic] to a density of 5x10^ cells/ml, and DNA extracted according to the methods g of Daphne Blumberg (personal communication). 5x10 c e l l s were washed 3 g times in KK2 buffer, pelleted, and resuspended to 5xl0'cells/ml, in the following lysis buffer: 10% sucrose, 0.5% NP40, 16 mM potassium chloride, 15 mM sodium chloride, 0.15 mM spermine, 0.5 mM spermidine, 15 mM fl-mercaptoethanol, 15 mM Tris-chloride (pH 7.4), 1 mM EDTA, 0.2 mM EGTA, and 1.5 mM phenylmethylsulfonylflouride. Cells were shaken vigorously in this buffer, pelleted at 4000 rpm for 10 minutes in a Sorvall GSA rotor, and the cloudy supernatant discarded. Nuclei were washed free of debris by two more washes in the same buffer, but without NP40. The nuclear pellet was resuspended in 15 ml sterile water, and adjusted to f i n a l concentrations of 0.1 M EDTA (pH 8) and 4% N-laurolsarcosine. Nuclei were lysed by gentle swirling at 50C. Sigma type XIII protease was added to a fi n a l concentration of 200 ug/ml, and the mixture was incubated for 2 hours at 37C with occasional swirling. The mixture was brought to 0.5 mg/ml ethidium bromide, and then cesium chloride was added to 0.905 gin/ml. This mixture was loaded into wide-mouth ultracentrifuge tubes, topped with parafin o i l , and spun at 26,000 rpm for 36 hours in a SW28 rotor. The protease treatment was necessary to allow the DNA to escape the protein at the top of the tube. A small pellet of RNA and debris was 24 visible at the bottom of the tube. The banded DNA was withdrawn with a Pasteur pipette inserted through the protein layer. After multiple butanol extractions, the DNA was dialysed for 36 hours in several changes of lx TE [10 mM Tris-chloride (pH 7.5) and 1 mM EDTA]. The DNA in solution was recovered from the dialysis tubing, and sodium acetate was added to give a fin a l concentration of 0.25 M. DNA was then ethanol precipitated overnight. The OD 260/280 ratio of the DNA obtained by this method was 2:1, and the DNA appeared to digest well with the restriction enzymes used. Southern blots. Hybridization of ras probes with digested genomic DNA from D.discoideum strain AX-3 was carried out as described in Southern protocol #4 provided in the Bio-Can Riboprobe l e a f l e t . Prehybridization was carried out at 50C for 4 to 6 hours in 50% formamide, 5x SSC, 20 mM sodium phosphate monobasic/sodium phosphate dibasic (pH 7.0), 0.5% SDS, and 0.05% Denhardt's reagent. Hybridization was carried out at 50C for a minimum of 16 hours in the same solution, but with the addition of 250 ug/ml denatured salmon sperm DNA, and 4x10^ cpm/cm2 of radioactively labelled RNA probe. Two 30 minute washes were carried out at 55C in 2x SSC, then one wash at 55C in 0.5x SSC, followed by one or two 30 minute washes in 0.2x SSC. Low stringency hybridizations employed the methods of Madaule et a l . (1984). Prehybridization was carried out for 8 hours at 42C in 30% formamide, 5x SSC, 5 mM EDTA, 0.1% SDS, 0.04% BSA, 0.04% F i c o l l , 0.04% polyvinlypyrolidine, 12 mM sodium phosphate monobasic/ 25 sodium phosphate dibasic (pH 7.0), 0.06% (w/v) pyrophosphate tetrasodium decahydrate and 100 /jg/ml salmon sperm DNA. Hybridization was carried out at 42C for 20 hours in 30% formamide, 5x SSC, 5 mM EDTA, 0.1% SDS, 0.04% Denhardt's reagent, 20 mM sodium phosphate monobasic/sodium phosphate dibasic (pH 7.0), 0.06% (w/v) pyrophosphate tetrasodium decahydrate, 50 ug/ml salmon sperm DNA, plus 4 x l o 5 cpm/cm2 of radioactively labelled RNA probe. Washes were carried out at 42C for 45 minutes each in 2x SSC, 2.5 mM EDTA, 0.1% SDS, 20 mM sodium phosphate monobasic/sodium phosphate dibasic (pH7.0), and 0.06% (w/v) pyrophosphate tetrasodium decahydrate. In vitro translation of D.discoideum RNA and immunoprecipitation of  products. Messenger dependant rabbit reticulocyte lysate (MDL) was made according to the method of Jackson and Hunt (1982). MDL was mixed with [35S]-methionine (Amersham) to give a fi n a l concentration of 2 juCi/ul. 30 ul of this mixture was added to dry RNA which was resuspended, and translated iri vitro for 2 hours at 30C. Incorporation of methionine was optimal with 2.2 to 3.8 /ug of total RNA/10 ul MDL, or with less than 0.4 ug of poly A+ RNA/10 ul MDL. After translation, 5 /ul of lysate was added to 1 ml water and 0.5 ml 1 M sodium hydroxide/1.5% (w/v) peroxide was added. The mixture was incubated for 15 minutes at 37C prior to the addition of 1 ml 20% trichloracetic acid (TCA). Precipitates were stored on ice for 20 minutes, and poured through 2.5 cm Whatman fiberglass GF/A f i l t e r s , and washed four times with 2ml 5% cold TCA. The remaining 25 jal 26 lysate was diluted with 75 ul of 259 lys i s buffer (0.5% SDS, 5 mM magnesium chloride, 100 or 400 mM sodium chloride, 20 mM Tris-chloride, 1% Triton X-100) plus 50 il l rabbit-anti-rat IgG-coated Staphylococcus ("Rarig Staph") resuspended to 10% (w/v) in the same buffer. After a 5 minute incubation at 4C, the mixture was centrifuged at 10,000 rpm for 2 minutes, and supernatants were transfered to fresh tubes containing 5 ug of Oncogene Sciences Y13-259 antibody. After overnight incubation on a tube rotator at 4C, 110 u l RaRIg was added to each tube for 2 hours and the mixture incubated for a further 2 hours. Finally, the immune complexes were centrifuged at 5000 rpm for 20 minutes, and washed 3x with 259 lysis buffer. The inclusion of 0.5% SDS in the 259 lys i s buffer reduced the total yield of ras protein in immunoprecipitates, but was essential to reduce backgrounds to acceptable levels. On advise from Dr. Virginia Chow, RaRIg without staph was used in immunoprecipitations, and vastly improved immunoprecipitation backgrounds. 27 RESULTS I) NORTHERN BLOTS OF DICTYOSTELIUM DEVELOPMENTAL TIME-COURSE RNAs. The probe sequence, subcloned into the SP64 transcription vector, was 4 a 470 bp Pst I fragment containing the coding sequence for amino acids 59 to 186 of the Dictyostelium ras protein. The fragment, kindly provided by Christophe Reymond, was part of the cDNA clone "Dd-rascl". 1) Measurement of level of total cellular ras RNA at different times  during development. Northern blots of total cellular D.discoideum RNA probed with Dd-rascl indicated that the 0.9 and 1.2 kb ras transcripts both appeared at 5 hours of development, and increased steadily until the last time point for which RNA was obtained, at 15 hours (Figure l a ) . Beneath the ras RNA doublets in lanes 3 to 7 of Figure l a , there was a t a i l of heterologously-sized ras transcripts ranging from 900 to 450 nucleotides long, decreasing in abundance with decreasing length. RNA degradation was the most lik e l y cause of this t a i l i n g beneath bands. A longer exposure of this Northern blot revealed the presence of low levels of ras mRNA in the RNA from 2 hour c e l l s , but no ras specific mRNA was detected in 20 ug of total RNA from vegetative c e l l s (data not shown). (However, low levels of ras mRNA were detected in 6 ug of poly A+ vegetative RNA, data not shown). The low level of ras mRNA at 0 and 2 hours is not li k e l y due to degradation by RNAses, since these early RNAs gave rise to abundant ras protein when _in vitro translated. While i t is possible that the ras RNA detected by the Dd-rascl probe does not encode the protein immunoprecipitated by the Y13-259 antibody, the fact that so much protein 28 Figure l a . Northern blot showing the developmental expression of D.discoideum ras in total RNA from strain V12M2, probed with Dd-rascl RNA. Total cellular RNA was phenol-chloroform extracted from developing cells at the times indicated. 20 jug of each RNA was glyoxalated and separated by electrophoresis in 10 mM sodium phosphate monobasic/ sodium phosphate dibasic (pH 7.0), through a 1.1% agarose gel. RNA was then blot-transfered to nitrocellulose. Hybridization with the Dd-rascl probe was carried out in 50% formamide at 55C for 16 hours, followed by three 20 minute washes at 65C in O.lx SSC, 0.1% SDS. Exposure was 2 days, with an intensifying screen. Figure lb. Northern blot with resolution of the two D.discoideum ras RNA species. Method as above, with the inclusion of one 20 minute wash at 80C. Lane contains 20 ng of total RNA from cells at 8 hours of development. 29 is synthesized from the 0 and 2 hour RNAs suggests their general integrity is at least equal to that of RNA samples from later time points. High stringency washes of Northern blots allowed resolution of the f u l l length bands of the ras doublet (figure l b ) . The sizes of the ras RNA bands detected in this work appeared to be the same as those reported by Reymond et a l . (1984), determined in the following manner. It has been noted that RNA probes may bind non-specifically to 18S and 28S rRNA in low stringency Northern blots (Promega bulletin, 1986). In this work rRNA migration was f i r s t checked by ethidium bromide staining of one lane of each gel to be used in Northern blotting. When bands appropriately sized to be rRNA were detected in low stringency Northern blots, they were used as internal size markers. Assuming linear migration of RNA species, the sizes derived for the two ras transcripts were consistent with those reported by Reymond et a l . (1984). In summary, Northern analysis of total RNA from c e l l s at different stages of development showed that the level of ras RNA in strain V12M2 increases steadily throughout the developmental program, rather than bimodaly, as suggested by the level of ras protein immunoprecipitated from lysates of c e l l s during development (Pawson et a l . , 1985) and by the level of ras RNA found in AX-3 cells-during development (Raymond et a l . , 1985). II) SOUTHERN BLOTS OF AX-3 DNA: DNA from strain AX-3 was used because the sizes of the ras genomic fragments in this strain were known (Reymond et a l . , 1984). The use of 30 DNA from axenically grown ce l l s also obviated artifacts seen in earlier Southern blots of DNA from bacterially grown V12M2 cells which possibly resulted from non-specific binding of the probe to bacterial sequences. 1) High Stringency Southern Blots Using the Dictyostelium ras probe: The bands detected by probing genomic DNA under conditions of high stringency with the Dictyostelium ras probe appeared to be the same sizes as those found by Reymond et a l . (1984) . The ras gene mapped to a 5.3 kb Eco RI restriction fragment, a 3.2 kb Eco RI-Bgl II restriction fragment, and to a Eco RV restriction fragment approximately 11 kb long (figure 2). The different intensities of binding were probably due to the better transfer of the shorter pieces of DNA. 2) Low Stringency Southern Blots Using the Dictyostelium ras probe: The same blot was then re-probed with the Dictyostelium ras sequence under conditions of low stringency, at 42C in 30% formamide (Madaule et a l . , 1985). Additional bands were indeed seen (figure 3). Their detection in this work, but not in low stringency Southern blots carried out by Reymond et a l . (1984), may have been fac i l i t a t e d by the large amount of DNA used per lane (15 ug), or because of the greater sensitivity of the probe system used. The Eco RV digested DNA showed faint bands in addition to the dominant 11 kb band, of 6.1 and 13.5 kb (lane 3). In undigested DNA (lane 1) the ras probe localized to a region of the blot corresponding to about 15 kb. In addition to the dominant band of 5.3 kb in Eco RI digests, three other faint bands were seen of 3.3 kb, 7.0 kb, and of about 9.1 kb (lane 2). In addition to the dominant band of 3.2 kb in Eco RI-Bgl II digests, three faint bands were seen of 1.75 kb, 5.5 kb, 31 9.4-6.7 4.4-2.3 2.0 Figure 2. Southern b l o t of AX-3 DNA, probed at high stringency w i t h Dd-rascl RNA. H y b r i d i z a t i o n s w i t h the Dd-rascl RNA probe were c a r r i e d out i n 50% formamide at 45C f o r 18 hours, then washed f o r 20 minutes once at 50C i n 2x SSC, once at 50C i n 0.5x SSC; then twice at 50C i n 0.2x SSC. Exposure was 20 hours, without a screen. 15 fig of DNA, d i g e s t e d w i t h the f o l l o w i n g r e s t r i c t i o n enzymes, was used per lane. Lane 1: undigested. Lane 2: Eco R l . Lane 3: Eco RV. Lane 4: Eco RI/Bgl I I . 32 1 2 3 4 probe= Dd- ras cl Figure 3. Southern b l o t of AX-3 DNA, probed at low st r i n g e n c y w i t h Dd-rascl RNA. As f o r f i g u r e 3. H y b r i d i z a t i o n was c a r r i e d out i n 30% formamide at 42C, f o r a minimum of 18 hours. The b l o t was washed twice f o r 30 minutes at room temperature and twice f o r 30 minutes at 42C, i n the s o l u t i o n s p e c i f i e d i n Methods f o r low s t r i n g e n c y Southern b l o t s . Exposure was 16 hours w i t h screen. 33 and of 9.1 kb (lane 4). Possibly the 9.1 Eco Rl fragment was not further digested by Bgl II, whereas the 7.0 and 3.3 kb Eco Rl bands gave rise to the 5.5 and 3.2 kb bands in the Eco RI-Bgl II digest by cleavage in 5' or 3' flanking regions. If so, this would suggest that the three Eco Rl bands represent at least two and possibly three non-contiguous ras-homologous sequences separated by many kilobases, therefore indicating the presence of two to three ras-homologous genes in addition to the one characterized by Reymond et a l . (1984). The faint bands detected in Figure 3 are not lik e l y artifacts caused by partial digestion, f i r s t l y because two of the bands are smaller than the dominant species seen under conditions of high stringency, and secondly because partial digestion products present in large enough quantities to be detected under conditions of low stringency, would be similarly detectable under conditions of high stringency. The optimum binding conditions for the high and low stringency bands were not established. It was however noted that when the same DNA blot used in Figures 2 and 3 was re-probed at hOC instead of kSC in 50% formamide, the dominant bands seen in Figure 2 plus only three of eight of the faint bands seen in Figure 3 were detected. These were the 7.0 kb band of lane 2, Figure 3; the 13.5 kb band of lane 3, and the 5.5 kb band of lane 4 (data not shown). 3) Southern Blots Of AX-3 DNA Using Viral ras Probes: Neither the Kirsten nor the Harvey probe hybridized to the Dictyostelium ras, or any other genomic sequence on a blot of digested AX-3 DNA. At the lowest stringency used, 42C in 30% formamide; blots had 34 high, even backgrounds (data not shown). As noted in the Methods, the HMSV and KMSV ras coding sequences share 82% homology (as determined by use of the Delaney SEQNCE program) in the f i r s t 100 base pairs of the ras coding sequence (the most highly conserved region) and cross hybridize under conditions of low stringency (Chang et a l . , 1982). In contrast, the Harvey and Kirsten ras sequences share only 69% and 75% homology respectively with the Dictyostelium ras sequence in the f i r s t 100 base pairs of coding sequence, as determined by use of the Delaney SEQNCE program. The Dictyostelium and HMSV sequences share one stretch of 11 base pairs of unbroken sequence homology in this region, and the Dictyostelium and KMSV sequences share one unbroken stretch of 14 base pairs. Other stretches of unbroken sequence homology are much shorter. In summary, new information from this work was primarily the detection by use of a D.discoideum ras probe at low stringency, of heterologous ras genomic sequences in D.discoideum strain V12M2 DNA. The results suggest the presence of two to three sequences heterologous to the probe sequence. Il l ) IMMUNOPRECIPITATION OF D.DISCOIDEUM RAS PROTEINS. In view of the differences in developmental gene expression detected in the studies of Reymond et a l . (1984) and Pawson et a l . (1985), RNA prepared from ce l l s at each developmental time point were translated in  vitro and immunoprecipitated, f i r s t l y to confirm that ras protein could be translated rn vitro from vegetative RNA despite the apparent absence of ras RNA in vegetative c e l l s , and secondly to see i f levels of ras protein were co-ordinate with the levels of ras RNA detected in Northern 35 blots. A number of intriguing and potentially important differences were found between the ras proteins synthesized _in vivo and in_ vitro. Comparison of in vitro translated ras proteins with in vivo synthesized  ras proteins. In cell-free lysates of strain V12M2, the dominant ras protein was 23 kd. p24 was a minor species not always seen jm vivo, but when present was most abundant in pseudoplasmodial cells at 14 hours. Although the pulse-chase labelling experiments required to establish the relationship between the _in vivo ras proteins were made d i f f i c u l t by the poor exchange between intracellular pools and exogenous methionine, particularly in older developing c e l l s , there was no evidence to suggest that p24 was a precursor to p23 in_ vivo (Weeks and Pawson, 198 ). In contrast, both p23 and p24 were synthesized _in_ vitro from the RNA isolated from V12M2 cells at a l l time points from vegetative stage to 15 hours of development. Furthermore, the 24 kd ras species was translated in equal or greater amounts than was the 23 kd ras species from the RNA from cells at a l l time points except 15 hours, when p23 frequently became the predominant ras protein species (figure 4). The different ratios of p23 and p24 obtained _in vivo and _in vitro may be due to the expression of the Dictyostelium RNA in a foreign translation system. To compare their sizes, ras proteins labelled jLn_ vivo were separated in polyacrylamide gels alongside ras proteins synthesized _in vitro (figure 5). As expected, only the p23 ras protein was present in c e l l s labelled in vivo during the f i r s t two hours of differentiation (lane 4). A lesser amount of p23 was present in cells labelled i_n vivo between 10 36 0 2 5 8 10 12 15 Figure 4. ras proteins immunoprecipitated from in vitro translations of developmental time-point RNAs from D.discoideum strain V12M2. Total RNAs from the indicated time points were translated for 2 hours at 30C in a messenger-dependant rabbit reticulocyte lysate (MDL) containing 2 juC/jul of [35s]-methionine. 5 jul was removed from each 30 jul reaction volume, trichloroacetic acid (TCA) precipitated and counted. 75 jul of 259 Lysis Buffer was added to the remaining 25 jul of each lysate, which were then "precleared" with 50 jul of 10% RaRIg Staph for 5 minutes as described in Methods. Each supernatant was then incubated with 5 jug of Oncogene Sciences Y13-259 or control antibody overnight on a rotator at 4C. 110 jul RaRIg was added to each tube for 2 hours. Immunoprecipitates were pelleted and washed three times in 259 Lysis Buffer. After electrophoresis through a 14% polyacrylamide gel, the gel was dried and autoradiographed. 37 Figure 5. A comparison of the rates of migration of ras proteins synthesized _in vitro and _in vivo. ras proteins were immunoprecipitated with the Y13-259 antibody from the following, and electrophoresed through a 16% polyacrylamide gel: Lanes 2_ and 3_: in vitro translated 2 hour RNA. Lane 2: no protease inhibitor added. Lane 3: 5 ug leupeptin added before and 5 ug added after translation. Lane 4^: cells labelled in vivo from 0 to 2 hours. } No protease in-Lane 5_: ce l l s labelled in vivo from 10 to 12 hours. } hibitors added. Lanes 6, 7, and 8_: _in vitro translated 12 hour RNA. Lane 6: 5 ug leupeptin added before and 10 ug after translation. Lane 7: 5 jug leupeptin added before translation. Lane 8: no protease inhibitor added. Lane 1: as Lane 2, but immunoprecipitated with Y13-238. Lane 9 : as Lane 8, but immunoprecipitated with Y13-238. 38 and 12 hours of differentiation (lane 5). The p22 breakdown product was evident in the latter experiment, but p24 was not detected. This was consistent with the findings of Weeks and Pawson (1987) . In contrast, both p23 and p24 were synthesized i_n vitro from the RNA of both 2 hour (lanes 2 and 3) and 12 hour cells (lanes 6-8) . The p23 ras protein synthesized _in vitro appeared slightly larger than the _in vivo p23 ras protein. This slight shift is likely due to the fatty acylation of the ras protein only _in_ vivo, which would reduce i t s apparent molecular weight in polyacrylamide gels (Buss et a l . , 1986). E l l i s at a l . (1981) found that the two murine Kirsten ras transcripts of 5.2 and 2.0 kb both produced the same size of p21 ras protein jjn vitro, and this protein also appeared slightly larger than the p21 protein seen jm vivo. The relationship between the two HI vitro ras proteins was f i r s t examined by testing the a b i l i t y of three protease inhibitors to alter the ratio of p23 to p24. Leupeptin and antipain were known to inhibit the degradation of p23 to p22 during the immunoprecipitation of ras proteins in Dictyostelium c e l l lysates (Weeks and Pawson, 1986) . These protease inhibitors were added to immunoprecipitations of _in vitro synthesized proteins in the event that Dictyostelium proteases synthesized in vitro had biological activity in rabbit reticulocyte lysates. When leupeptin (figure 5; lanes 3,6 and 7) and antipain (figure 6, lane 1) were included prior to translation of the RNA, or PMSF prior to the overnight incubation of the proteins with Y13-259, (figure 6, lane 2) these protease inhibitors had no effect on the ratio between the two ras proteins. However, both antipain and PMSF reduced the amounts of two 39 1 2 3 4 Figure 6. Effect of protease inhibitors on ras proteins translated J_n vitro. Lanes 1, 2, and 3_: ras proteins immunoprecipitated from i_n vitro translation reactions of total 12 hour RNA. Lane 1: 5 jug leupeptin added at outset of translation, and 10 jug added at outset of immunoprecipitation. Lane 2: 10 ug phenylmethylsulphonylf lor ide (PMSF) added at outset of immunoprecipitation. Lane 3: no protease inhibitors added. Lane 4_: ras proteins immunoprecipitated from AX-3 cells labelled in  vivo from 0 to 2 hours of development. No protease inhibitors added. Proteins were separated by electrophoresis through.a 16% polyacrylamide gel. 40 minor degradation products of 21 and 20 kd. The failure of a l l three protease inhibitors to effect the ratio of p23 to p24 suggests either that a degradation takes place which is not sensitive to these inhibitors, or that p23 is not derived by proteolysis from p24. To further examine the relationship between the two ras proteins, RNA was translated in_ vitro for increasing lengths of time, to allow the detection of changes in the ratios of p23 to p24 in immunoprecipitates with increasing incubation time. An accumulation of the smaller protein over time would indicate that post-translational modification occurs in vitro, whereas a constant ratio would provide evidence that neither protein is derived from the other. This latter possibility seems to be the case. A constant ratio of the two proteins was observed between 10 and 120 minutes of translation of each RNA. Both proteins are translated in roughly equal amounts from vegetative RNA (figure 7), the larger species predominates in 2 hour RNA (figure 8), equal amounts are seen in 12 hour RNA (figure 9), and the smaller species is synthesized in larger quantities in 15 hour RNA (figure 10). These results suggest the two proteins are not derived from one another . I.f true,they must be translated from separate mRNAs. It cannot be determined from this work whether these two mRNAs are derived from the same gene (by ut i l i z a t i o n of alternate splice sites, or translational stop sites), or from two separate genes. Amount of ras protein expressed throughout the developmental program: A quantitation of the amount of p23/p24 synthesized in vitro was attempted, to better compare the amount of ras protein synthesized in 41 T=0 1 0 ' 3 0 ' 6 0 ' 1 2 0 ' c 66-424 «23 14-Figure 7. Ratio of ras proteins seen when vegetative RNA was translated ir± v i t r o for increasing lengths of time. RNA was translated i n a 300 u l volume, and at the times indicated aliquots were removed f o r immunoprecipitation with 5 ug Y13-259 antibody. A 125 u l a l i q u o t , rather than the usual 25 u l , was removed at 10 minutes to compensate for the lesser amount of incorporation i n t h i s early time point (about 25% of the f i n a l incorporation at 120 minutes), and inadequate precle a r i n g may have caused the high backgrounds seen. Lane "c" (cont r o l ) : a 25 u l aliqu o t of the t r a n s l a t i o n mixture was removed at 120 minutes and immunorecipitated with Y13-238 antibody. Proteins were separated on a 16% polyacrylamide g e l . 42 1=2 10' 30' 60' 120' C 4 24 «23 Figure 8. Ratio of ras protiens seen when 2 hour RNA was translated in  v i t r o for increasing lengths of time. 2 hour RNA was translated in a 300 jal volume, and at the times indicated, 25 nl a l iquots were removed for immunoprecipitation with 5 jag Y13-259 antibody, except for lane " c " (control) which was a 25 >ul a l iquot removed at 120 minutes and immunoprecipitated with Y13-258 antibody. No protease i n h i b i t o r s were included. Proteins were separated on a 16% polyacrylamide g e l . 4 3 Figure 9. Ratio of ras protiens seen when 12 hour RNA was translated in  vitro for increasing lengths of time. 12 hour RNA was translated in a 300 jul volume, and at the times indicated, 25 u l aliquots were removed for immunoprecipitation with 5 jug Y13-259 antibody. "+" indicates the addition of 5 /ug leupeptin immediately after translation. "-" lanes had no protease inhibtor added. Lane "c" (control) was a 25 jul aliquot removed at 120 minutes and immunoprecipitated with Y13-258 antibody. Proteins were separated on a 16% polyacrylamide gel. 44 T=15 1 0 ' 3 0 ' 6 0 ' 1 2 0 ' c Figure 10. Ratio of ras protiens seen when 15 hour RNA was translated i vitro for increasing lengths of time. 15 hour RNA was translated in a 300 jul volume, and at the times indicated, aliquots were removed for immunoprecipitation with 5 ug Y13-259 antibody. A larger aliquot, 125 u l rather than the usual 25 u l , was removed at 10 minutes to compensate for the lesser amount of incorporation in this early time point, as in figure 12. Lane "c" (control) was a 25 u l aliquot removed at 120 minutes and immunoprecipitated with Y13-258 antibody. Proteins were separated on a 16% polyacrylamide gel. 45 vitro to the levels of ras RNA detected in Northern blots- ras proteins were translated _in vitro from the total cellular RNAs used in the Northern blot shown in figure 1, immunoprecipitated from translation reactions with the monoclonal antibody Y13-259; then separated on SDS polyacrylamide gels (as shown in Figure 4) and quantitated. Quantitation of total ras protein per translation reaction involved s c i n t i l l a t i o n counting of p23/p24 bands that had been excised from polyacrylamide gels. The counts per minute found in each ras doublet was divided by the counts per minute of [35S]-methionine incorporated into 5 ul of the _in_ vitro translation reaction of each RNA. A plot of the relative amount of ras protein synthesized _in vitro from each RNA in two separate experiments (Figure 11) shows that the total amount of ras protein synthesized in the rabbit reticulocyte system was greatest in protein translated from 2 hour RNA. The second greatest amount of ras protein was translated from vegetative RNA, and lesser amounts of ras protein were translated from the RNA of cells 5 to 15 hours into development (Figure 11). RNAs from early and late cells incorporated [35S]-methionine equally well in the in  vitro translation reactions, however the absolute amount of incorporation varied from one experiment to another. Some loss of total ras protein resulted from stringent wash conditions and from "pre-clearing" with rabbit-anti-rat immunoglobulin-coated Staph. These steps were necessary to reduce immunoprecipitation backgrounds (data not shown). Nonetheless, the two experiments plotted in figure 11 were internally consistent with each other. In summary, the pattern of expression of ras appears different for 46 H o u r s o f d e v e l o p m e n t Figure 11. Plot of the relative amount of [35S]-methionine incorporated into p23/p24, relative to the total protein synthesized in  vitro from the RNA of developing strain V12M2 c e l l s . Experiments were carried out as described in Methods. 5 jul of each 30 >ul reaction were removed after 2 hours translation at 30C, and the [35s]-methionine-labelled protiens were precipitated with trichloroacetic acid, washed and s c i n t i l l a t i o n counted. The remaining 25 jul of translation mixture was immunoprecipitated with 5 ug Y13-259 antibody. Immunoprecipitates were washed, resuspended in lx SDS sample buffer, and separated by electrophoresis through 12.5% polyacrylamide gels. The combined p23/p24 bands were excised and sc i n t i l l a t i o n counted, and their percentage counts per minute (cpm) relative to the total cpm in immunoprecipitation volumes determined. 47 Northern blots of whole RNA (figure 1) and for ras proteins synthesized in vitro from whole RNA (figure 11). The predominant difference is that the greatest amount of ras protein is synthesized in_ vitro from the RNA of early developing c e l l s , whereas the greatest amount of ras RNA is present in 12 and 15 hour c e l l s . It is possible that vegetative and early ras mRNA is translated with very high efficiency, whereas later RNA, present in far greater quantities, is not. A second explanation for the abundant amount of ras protein in vegetative cells is that the Dd-ras cl probe and the Y13-259 antibody do not recognize the same gene products. Or perhaps the early abundant p24 is encoded by a second ras gene not detected by the Dd-rascl probe. The pattern of proteins translated from the RNA of developing c e l l s in  vitro is also different from the pattern of ras protein immunoprecipita-ted from lysates of developing c e l l s . The expression of the latter is essentially bimodal, with the larger peak of ras synthesis occurring in early c e l l s , and the smaller peak occurring in 12 hour c e l l s (Pawson et a l . , 1984). The reason for the absence of the second peak of ras when RNA is translated rn vitro is unknown. The two ras proteins in D.discoideum appear not to be related to one another by degradation or post-translational processing, suggesting they are encoded from two different mRNAs, which may be derived from either one gene or two. 48 DISCUSSION It was of interest to compare the results of Northern blots done in this work to those of Reymond et a l . (1984) who originally cloned the D.discoideum ras gene and published the f i r s t Northern analysis of i t s developmental expression. They analysed poly A+ RNA only, from strains AX-3 and NC-4. A 1.2 kb ras transcript was detected in variable amounts in AX-3 vegetative c e l l s . Ras RNA was not detected again until 15 hours of development when both the 1.2 kb and a 0.9 kb species appeared, then increased to a maximum at 17 hours, and declined to zero by culmination. In the present work using strain V12M2, Northern blots of total RNA show that transcription of the 0.9/1.2 kb ras doublet increases steadily from 5 hours until 15 hours (Figure la) (with very low levels of ras RNA present at 2 hours and even less at 0 hours). The lack of concurrence between the results of Reymond et a l . (1984) and the present work may be due to strain differences. Reymond et a l . (1984) found strains AX-3 and NC4 to differ in that NC4 lacked any early ras expression, so that the ras doublet f i r s t appeared at 12.5 hours. The strain used in the present work, V12M2 may differ from both AX-3 and NC4. A similar result to that of Reymond et a l . (1984) for NC4 was found in the present work, except that low levels of the smaller ras species were detected in total NC4 RNA from 0 hours of development until the onset of pseudoplamodial stage, when much greater amounts of the ras doublet appeared. The detection of the early ras RNA in NC4 may be due to the greater sensitivity of detection obtained with the RNA probe (data 49 not shown). The pattern of ras RNA expression differed from the pattern of expression of ras protein seen in_ vitro and _in_ vivo. The greatest amount of protein was synthesized hi vivo at 0 and 2 hours of development, when the least amount of ras RNA is detected, and a substantial but lesser peak of ras protein synthesis occurred iri vivo at 15 hours (Pawson et a l . , 1985) when the greatest amount of ras RNA was detected in Northern blots in the present work. The greatest amount of ras protein was translated _in vitro from the RNA of 2 hour ce l l s , large amounts were translated from the RNA of vegetative cells, and relatively l i t t l e from latter stages of development. It was not understood why the second peak of ras protein synthesis was absent _in vitro, since radioactive label incorporation was comparable to that of RNA from earlier time points. There are several explanations for the lack of correlation between ras RNA levels detected using the Dd-rascl probe and the amount of ras protein immunoprecipitated from in vitro translations by the Y13-259 antibody. F i r s t l y , the efficiency of translation of the ras mRNA might vary greatly throughout development, such that the small amount of RNA present in early developing cells is highly actively translated, whereas the abundant ras RNA present at pseudoplasmodial stage is translated very poorly. Secondly, the Dd-rascl probe and the Y13-259 antibody may recognize the products of different genes. Thirdly, perhaps the ras RNA detected by the Dd-rascl probe gives rise to a portion of the ras protein immunoprecipitated by the Y13-259 antibody, but a second gene not 50 detected by the Dd-rascl probe and actively translated in vegetative cells, and encodes the greater portion of the early stage ras protein, possibly the more abundant p24. Interestingly, a 23 kd and 24 kd ras protein were synthesized both in  vivo and In vitro, but their relative proportions and patterns of expression were radically different in these systems. The major species throughout development _i_n vivo is the 23 kd protein, and the minor 24 kd species, when seen, is most abundant at pseudoplasmodial stage (Weeks and Pawson, 1986). In vitro, the major species translated from the RNA of cells from every time point except 15 hours was 24 kd (figure 4). Tryptic peptide digests of D.discoideum ras proteins were carried out previously by Pawson et a l . (1985). The pattern of [35S]-methionine-containing peptides separated by two dimensional electrophoresis were very similar for p23 _in_ vitro, p24 _in vivo, and p23 _in vivo, although the f i r s t two species appeared to be the most closely related to one another. Results of _in vitro translation of V12M2 RNA in this work suggest that p23 and p24 are not related by post-translational processing or by degradation (figures 7 to 10). An apparent constant ratio of p23 to p24 was translated _in vitro from each RNA tested, as determined by removing a sample of the proteins synthesized at intervals of a two hour _in vitro translation. Alternatively, this data could result from a precursor/ product relationship between p23 and p24, providing the rate of formation of the precursor was equal to the rate of decay of the product protein. This seems possible, since the h a l f - l i f e of p23 (reported to be 2.35 +/-51 0.07 hours in early developing strain AX-2 cells)(Weeks and Pawson, 1987) would result in a significant loss of p23 over the 2 hour translation period. However, the protein processing would have to occur only during in vitro translation, otherwise, levels of the product protein would accumulate during the overnight immunoprecipitation on ice. If the two ras proteins of strain V12M2 are derived from separate mRNAs, these mRNAs may be derived from two separate genes which are expressed differentially throughout development, or the two RNAs may be derived from the same gene by alternative splicing or perhaps by alternate transcriptional start sites, such as seen in the Drosophila alcohol dehydrogenase gene (Benyajati et a l . f r 1983). The latter possibility requires that these alternate sites be utilized in different proportions in vitro than ijn vivo, since the relative amounts of p23 and p24 dif f e r in vitro and iri vivo. The relationship between the two ras proteins and the two ras RNA species detected.is presently unknown. It seems possible that p24 may be derived from the the 1.2 kb ras transcript, and p23 from the 0.9 kb transcript. However, this model cannot apply to the early stages of development, when very low (or zero) levels of just one ras RNA species is detected, yet large amounts of p24 and p23 are translated from the same RNA stocks _in vitro. This suggests either that a second ras transcript exists undetected in early stage RNA, or as previously suggested, that the RNA probe and the Y13-259 antibody do not recognize the same gene products. 52 The human Kirsten ras gene gives rise to two mRNAs by alternate splicing of the 3' exons "4A" and "4B", and the protiens encoded by these RNAs are slightly different in size (McGrath et a l . , 1983). By analogy, both of the Dictyostelium ras proteins may be derived from the one known ras gene i f i t contains alternate splice sites within the known exons, or i f a hitherto undiscovered exon exists outside of the sequenced region of the ras gene. This hypothesis also requires that the ratio of alternate splice sites u t i l i z e d changes during development. The unusually high levels of p24 _in vitro may be aberration caused by translation of Dictyostelium RNA in a rabbit reticulocyte lysate. In addition to the expected D.discoideum ras restriction fragments, additional ras-homologous bands were detected in genomic AX-3 DNA under conditions of low stringency (30% formamide, 42C). The bands may represent more than one heterologous ras genomic sequence. Transcripts derived from these heterologous ras sequences should be detectable in Northern blots under similar hybridization conditions. No new ras-homologous RNAs were detected in low stringency Northern blots of vegetative RNA to account for the large amount of protein synthesized at this stage. Hence, there is at present no good evidence to suggest that the new ras-homologous genomic sequence is transcriptionally functional. An interesting correlation was noted between the peaks of ras protein expression, and the peaks of mitotic activity that occur during the developmental program. Zada-Hames et al.(1978) found that two peaks of mitotic activity occurred during the D.discoideum developmental program, 53 centered over 4 and 16 hours. Furthermore, [3H3-thymidine uptake assays indicated that two periods of DNA synthesis also occurred during development, and coincided with the two periods of mitosis. It was estimated from autoradiographs of disaggregated fixed c e l l s , that only 25% of cells incorporated 3H-thymidine in the f i r s t period of mitosis/DNA synthesis, during which 47% of the c e l l population underwent mitosis; and only 10% of cells incorporated 3H-thymidine in the second period, at 16 hours, when 25% of the c e l l population was undergoing mitosis. Pawson et a l . (1985) found two peaks of ras protein expression in_ vivo, centered over 1 and 12 hours, with the peak at 1 hour always greater than the peak at 12 hours. Thus during development, a peak of ras protein expression precedes by about 3 hours, each peak of mitosis and DNA synthesis. 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