isolation and characterization of transfer rnas from dicty during growth and development

7/30/2019 Isolation and Characterization of Transfer RNAs From Dicty During Growth and Development

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C M Palatnik and E R Katzduring growth and development.RNAs from Dictyostelium discoideumIsolation and characterization of transfer:

1977, 252:694-703.J. Biol. Chem.

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Isolation and Characterization of Transfer RNAs fromDictyosteZium discoideum during Growth and Development*(Received for publication, August 13, 1976)

CARL MATHEW PALATN IK~ AND EUGENE R. KATZFrom the Department of Cellular and Comparative Biology, State Unive rsity of New York at StonyBrook, Stony Brook, New York 11794MICHAEL BRENNERS:From the Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

Transfer RNAs and aminoacyl-tRNA synthetases wereisolated from vegetative and developing cells of the cellularslime mold Dictyostelium discoideum. Using the homolo-gous synthetases, the tRNAs were compared for their levelsof acceptance of 17 amino acids. These levels were found tobe the same. Th e tRNAs were further compared by doublelabel chromatography on reversed phase columns. Somemajor quantitative differences in individual peaks were ob-served. In addition, for a number of aminoacyl-tRNAs,small but reproducible displaceme nts of individual peakswere apparent. In many cases the displacemen t was in themajor peak, and in all but one case the developmental peakeluted ahe ad of the vegetative peak. Taken together, theseresults suggest that there i s no change from growth todevelopment in the transcription of tRNA genes, but thatthere are important changes in post-transcriptional modifi-cation.

Differences in chromatographic mobilitie s have been dem-onstrated for tRNAs from a wide variety of tissues in differentmetabolic or developmental states (for detailed reviews seeRefs. 1 and 2). Since the existence of such differences is quitecommon, an understanding of their nature and functionalsignifican ce is of general biologica l interest. In some highlyspecialized differentiating systems, tRNAs have been shownto adapt to the amino acid composition of the few major pro-teins being synthesized (3-61, and therefore appear to be com-ponents of the differentiation process itself. In a few of thesecases there is also some evidence that the availability of a fewtRNAs may limit and thereby regulate the rate of synthesis ofthese proteins (5, 7). However, most changes in metabolic ordevelopmental state do not involve gross changes in the aminoacid compos ition of the proteins being made. In these morecommon instances it has proved difficult to proceed beyondobservations of correlations between changes in tRNAs and

* This work was supported by grants to E. R. K. from the UnitedStates Pu blic Health Service (GM 18476) and to M. B. from theMilton Fund, Clark Fund, and the National Science Foundation (GB32042 and BMS 72.01830 A02).$ Present address, Department of Microbiology, University ofMassachusetts Medical School, Worcester, Mass. 01605.

5 To whom correspondence should be addressed.

changes in the biologic al state of cells to the goal of under-standing cause and effect relationships. Only in a few studieswith bacteria has it been possible to ascribe functions to thetRNA changes. In Salmonella typhimurium a tRNA modifica-tion has been found essential for proper transcriptional controlof several amino acid biosynthetic operons (8, 9). For technicalreasons, eukaryotes have not been as amenable to analysis asbacteria. Except for the highly specialized systems notedabove, the significan ce of tRNA changes in eukaryotic systemsremains unknown.

Among eukaryotic organisms, the cellular slim e mold Dic-tyostelium discoideum appears to be especially promising forpursuing an analysis of developmental changes in tRNAsbeyond the stage of simple correlation. In addition to its ease ofmanipulation and the fact that it differentiates in 24 h intoonly two cell types, the technology exists for assaying for thetranscriptional and translational consequences of any detectedalterations. Wheat germ and rabbit reticulocyte cell-free pro-tein-synthesizing systems have been shown to be extremelyefficient in translating stage-specific Dictyostelium mRNAs(10). Furthermore, an in vitro system for synthesizing mRNAprecursor, using isolated nuclei, has been developed and hasbeen shown to give excellent initiation (111. Finally, thehaploid nature of the organism facilitates obtaining mutants(12, 13). The utilization of mutants to elucidate the role oftRNA in the repression of certain amino acid biosyntheticoperons in bacteria (14) indicates the utility of this feature.

We are interested in the possibility that tRNAs might beinvolved in regulation of either translational or transcrip-tional events during development of D. discoideum. Becauseof the well known role of tRNA in decoding the base sequenceof mRNA during protein synthesis, considerable attention hasbeen given to the possibility that tRNAs may be involved inregulating the translation of messenger RNAs (1, 2). But inaddition to their part in translation, tRNAs act also to donateamino acids to lipids, peptidoglycans, and proteins (15); and inbacterial systems at least, have been implicated in the tran-scriptional regulation of certain amino acid biosynthetic path-ways (8, 9, 14). Hence tRNAs may assume multiple roleswithin an organism, including functioning as regulatoryagents at several levels of control.

I C. T. Mabie and A. Jacobson, personal comm unication.694

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tRNAs o/Uictyoste lium discoideum 695We have become intrigued by the recent finding that starva-

tion for certain amino acids is apparently required for initiat-ing development (16). Changes in in uiuo levels of tRNAaminoacylation could conceivably alter the protein syntheticpatterns in early development. In addition, by analogy withthe role of tRNAs in expression of certain amino acid biosyn-thetic operons in bacteria (8, 9, 141, the signal for amino acidstarvation and consequently the signal for initiation of devel-opment in Dictyostelium may well be communicated to thetranscriptional control apparatus by tRNAs.

In order to provide a broad framework from wh ich to initia tea functional analysis of tRNAs in Dictyostelium development,a general characterization of tRNAs present during growthand 18 h of development (initiation of culmination) was made.This developmental stage was chosen as it allowed a maxi-mum of time for changes in tRNAs and aminoacyl-tRNAsynthetases to accumulate. Still later times were not satisfac-tory because during culmination stalk cells extrude their con-tents, vacuolate, and die. Following the isolation of tRNAsand aminoacyl-tRNA synthetases, we have determined thereaction conditions for optimum aminoacylation of the tRNAswith each of 17 amino acids, and this has in turn enabled us toassay for quantitative changes in acceptance for each of theseamino acids during development. Finally, we have analyzedisoaccepting species of aminoacyl-tRNAs using reversed phasecolumn chromatography. The combined results of these stud-ies suggest that during Dictyostelium development tRNAs areregulated primarily at the level of post-transcriptional modifi-cation.

MATERIALS AND METHODSCulturing and Harvesting of Cells-Dictyostelium discoideum

strain A3 (17) was grown at 22-23 on a gyratory platform shakerrotating at 175 to 200 rpm. Cel ls were grown in HL-5 mediumcontaining yeast extract (Difco), proteose peptone (Difco), glucoseand phosphate buffer at pH 6.5 (18, 19). Cultures containing 2 litersof medium were routinely grown in 4-liter flasks and had a genera-tion time of 9 to 10 h. Exponentially growing cells at concentrationsof less than 6 x 10 cells/m l were harvested in a Sorvall RC-2Bcentrifuge at 1500 x g for 30 s at 4 or in a Sharples continuous flowcentrifuge.

Plating Cells for Development and Haruesting of DevelopingCells -Exponentially growing cells were harvested, washed once ata titer of 1 x lO/ml with PDF b uffer (40 rnM KCl, 10 rnM MgCl,, 18.5mM K,HPO,, 26.5 mM KH,PO,, and 500 kg/ml of streptomycinsulfate, pH 6.4 to 6.61, and resuspended in PDF buffer at a finalconcentration of 3 x 10 cells/m l. Five milliliters of the cell suspen-sion were evenly distributed on a 12.5~cm Whatman No. 50 filter,supported by eight Whatman No. 3 12.5~cm filters in a l&cm Petridish. The filters had been previously saturated with 40 ml of PDFbuffer and freed of air bubbles with a glass spreader. Petri disheswere placed in polyethylene bags on flat trays containing moistpaper towels. Bags were sealed with tape and incubated at 22. After18 h of development, just before culmination commenced, cells werescraped from the filters and washed once. Details of wash buffers aredescribed in subsequent sections. Only cells with an apparent syn-chrony of greater than 80% were used.

Preparation of tRNA -Transfer RNA w as prepared by a mod iflca-tion of a method originally utilized for isolation of tRNA from Salmo-nela typhimurium (20). Transfer RNAs from exponen tially growingcells (vegetative tRNA) and from cells at 18 h of development (devel-opmental tRNA) were isolated in identical fashion. It was essentialthat freshly harvested cells be used for the tRNA preparations, asfrozen cells yielded material of extremely low purity.

Cells were harvested, washed once with TMS buffer (10 rnM Tris/HCl, pH 7.5, 10 rnM MgCl,, and 2 mM Na,S,O,) and resuspen ded inTMS buffer at a final concentration of 2 ml/g of cells. A n equalvolume of pheno l (Mallinckrodt), saturated with TMS buffer, wasadded and the mixture was shaken vigorously for a minimum of 1 hat 4. The resulting suspension was centrifuged at 23,000 x g for 30

min. T he aqueous phase was withdrawn, 1150 volume of 5 M NaCladded to give a final concentration of 0.1 M NaCl and 2.5 volumes of95% ethanol added. The solution was stored at -20 for up to a monthwithout any deleterious effects, during which time other phenolextracts were prepared and combined with it. As much ethanol aspossible wa s removed with an aspirator and the remaining precipi-tate collected by centrifugation at 16,000 x g for 30 min. The pelletwas dried under vacuum, resuspended in 0.02 M NaCl in TMS buffer,and extracted twice with an equal volume of ether saturated with thesame buffer. The resulting solution was passed onto a DEAE-cellu-lose column (Whatman DE52, l-m l bed volume/g of cells) previouslyequilibrated with 0.02 M NaCl in TMS buffer. The column waswashed at a flow rate of 2 to 5 mlimin until the A,,, of the effluentwas about 0.1 and then with 0.1 M NaCl in TMS buffer until the A,,,was about 0.05. The tRNA was eluted with 1.0 M NaCl in TMS bufferat a flow rate of 1 mlimin . All fractions with an A,,, of greater than1.0 were pooled and the tRNA precipitated by addition of 2.5 volumesof 95% ethanol followed by overnight storage at -20. The precipitatewas collected by centrifugation at 16,000 x g for 30 min. The precipi-tate was dried under vacuum, redissolved in 1.8 M Trisiacetate, pH8.0, and any attached amino acids removed by incubation at 37 for90 min (21). Ice-cold 95% ethanol (2.5 volumes) was added and thetRNA precipitate kept at -20 for at least 15 min to ensure completeprecipitation. The precipitate was collected by centrifugation at27,000 x g for 15 min, washed twice with 75% ethanol containing 0.1M NaCl, dried under vacuu m at room temperature, and stored as apowder at -20.

Th e A&APHO of tRNA prepared in this manner was 2 and theaverage yield was about 1 mgig of cells. T he A,,,/mg of tRNA in abuffer of 100 mM sodium cacodylate, pH 7.5, 10 mM MgCl,, and 2 mMNa&O, was 15. The tRNA preparations contained about 5% proteinas determine d by the biuret method (22) and les s than 0.1% primaryaldehydes (which include DNA) as measured by the diaminobenzoicacid assay (23). There were no apparent differen ces in these proper-ties for vegetative and developmental tRNA preparations. Electro-phoresis on polyacrylamide gels showed the tRNA preparation to beabout 10% pure before the DEAE -cellulose chromatography and 50 to60% pure at the final step, the primary contaminant being ribosomalRNA. This estimate of purity agrees well with the results fromaminoacylation (Table II). Assum ing that alanine, glutamine, andcysteine are accepted by the preparations to the same extent as theaverage of the other 17 amino acids, a total of about 1060 pmol ofamino acids were accepted per A,,, unit of tRNA (average for thevegetative and developmental preparations shown in Table II). Sincepure tRNAs accept about 1700 pmol of amino acids/A,,,, unit (241, onthe basis of amino acid acceptance our preparation was also about60% pure.

Preparation of Aminoacyl-tRNA Synthetases - Aminoacyl-tRNAsynthetases from exponentially growing cel ls (vegetative synthe-tase) and from cells at 18 h of development (developmental synthe-tase) were prepared in identical fashion.Cells were harvested, washed once at a titer of 1 x lOHim withcold synthetase buffer (10 mM TrisiHCl, pH 7.5, 10 mM MgCl,, 10%glycerol, and 14 mM mercaptoethanol), and resuspended at a final

concentration of about 0.8 to 2 x lo9 cells/m l. Cells were homoge-nized with a tight fitting Dounce homogenizer until 90% breakagewas achieved. Lower cel l concentrations resulted in less efficientbreakage. Alternate procedures for cel l breakage (sonica tion, freeze-thawing, detergent lysis) gave much poorer activities.The A,,, of the homogenate was adjusted to about 80 and thehomogenate centrifuged at 37,000 x g for 30 min at 4. The clearsupernatan t was removed, transferred to an ultracentrifu ge, andcentrifuged at 105,000 x g for 1 h. Failure to dilute the cell extractoccasionally yielded preparations of reduced activity, probably be-cause particulate material remaining in suspension interfered withthe DEAE-ce llulose chromatography which followed.

The supernatant from the 105,000 x g centrifugation step waswithdrawn and l/q volume of 1 M KC1 was added to give a finalconcentration of 0.2 M KCl. It was then p assed onto a DEAE-cellu-lose column (Whatman DE52, 6-ml bed volume/g of cells), which hadbeen previously equilibrated at 4 with buffer containing 0.2 M KCl,50 mM TrisiHCl, pH 7.5, 10 mM MgCl,, 10% glycerol, and 14 rnMmercaptoethanol. It was eluted directly with the same buffer. Frac-tions contammg an ALGO/APHO of les s than 1.2 (greater than 95%protein) were pooled. The pooled fractions were concentrated andfree amino acids were removed by vacuum dialysis against twochanges of synthetase buffer, the second containing 50% glycerol.



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696 tRNAs of Dictyoste lium discoideumThe first dialysis was for a minimum of 2 h against at least 10volumes of buffer and the second was for a minimum of 4 h against atleast 200 volumes of buffer. Alternatively, the pooled column ef-fluent was brought to 85% saturation with enzyme grade ammoniumsulfate (SchwarziMann) and the resulting precipitate was dialyzedagainst two changes of at least 200 volumes of synthetase buffer forat least 2 h each before being brought up to 50% glycerol. The columnstep was necessa ry both to remove tRNA from the enzyme prepara-tion and to eliminate some component(s) which reduced both the rateand extent of the aminoacylation reactions. This component was notremoved by either ammonium sulfate precipitation or dialysis. Somepreliminary experiments eliminated trivial explanations for the in-hibition: it did not result from simple exhaustion of ATP, am ino acid,or enzyme, or from comp lexing of MgCl,. Nor did it appear to be dueto degradation of tRNA or accumulation of pyrophosphate or 5-AMP. The nature of the inhibition was not further pursued.Synthetas e preparations were stored at -20 and were stabl e forat least 6 months. Final protein concentrations (2.51 were 25 to 30 mgiml in preparations concentrated with ammonium sulfate and 50 to 65mgiml in preparations concentrated by vacuum dialysis. Incubationsin aminoacylation reactions with the tRNA omitted gave very lowbackground counts, indistinguisha ble from reaction mixtures lack-ing both synthetase and tRNA, indicating that synthetase prepara-tions were not contaminated with tRNA. No tRNA degradativeproperties were detected in synthetase preparations during amino-acylation reactions, as all aminoacylation reactions reached andmaintained reliable platea us which were unaffected by preincubat-ing the tRNAs with synthetases before adding labeled amino a cid.Chromatography on reversed phase columns also indicated an ab-sence of degradative activities in the synthetase preparations. Thechromatographic profiles showed sharp peaks and were highly repro-ducible regardless of the synthetase preparation used for the amino-acylation. In addition, vegetative tRNA aminoacylated with vegeta-tive synthetase for 5 min with 1Hlleucine and vegetative tRNAaminoacylated with vegetative synthetase for 60 min with[Clleucine, gave identical profiles when co-chromatographed.

Optimization of Assay Conditions - Assays were performed at 22since this is the temperature for optimal growth ofD. discoideum. (Afew assays conducted at 37 gave no activity). The buffer and pHwere selected by optimizing for the acceptance of leucine by thetRNA. Cacodylate buffers gave levels of incorporation higher thanbuffers of phosphate or Tris, and a pH of 7.5 was found superior to6.5, 7.0, and 8.0. Other reaction components were individually op-timized for each of the amino acids, using the following procedure. Astandard 50 ~1 of assay mixture contained 100 mM sodium cacodyl-ate, pH 7.5, 5 mM ATP, 10 mM MgCI,, 10 pM IH-aminoacid, 50 FMconcentration each of the other 19 unlabeled amino acids (except thatasparagine, glutamine, and glutamic acid were omitted from theaspartic a cid reaction mixture; glutamine and asparagine from theglutamic acid mixture; and glutamine and glutamic acid from theasparagine mixture) and about 2.5 A,,;,, units of vegetative tRNA.Reactions were terminated by precipitation on filter paper disc s with5% trichloroacetic acid according to the procedure of Rubin et al. (26)and counted in a toluene-based scintilla tion fluid containing 4 g of2,5-diphenyloxazole (PPO) and 0.1 g of 1,4-bisl2-(5.phenyloxa-zolylllbenzene (POPOPJiliter of toluene. The first step in the optimi-zation procedure was to vary the conce ntration of the vegetativesynthetase. The concentration which yielded and maintained thehighest plateau level was chosen for subsequent assays. The concen-tration of the synthetase was one of the most critical variables andhad to be carefully determine d for each enzyme p reparation. Concen-trations which were too low reduced both the rate and extent of thereaction, whereas concentrations which were too high did not main-tain a plateau value. Similar observations have been made in othersystems (27).

After the optimum enzyme concentration was obtained, the con-centration of the H-aminoacid was varied until the plateau was nolonger dependent on the amino acid concentration. Finally, theMgCl, and ATP concentrations were varied both together and inde-pendently. Once the reaction components were optimized, the reac-tion was tested to ascertain if the incorporation was linear withtRNA concentration.

Preparation of Aminoacyl-tRNA for Columns -Following amino-acylation of the tRNA from both stages under cond itions which gaveoptimum aminoacylation, the reaction mixes from each stage weremade 0.01 M with acetic acid to reduce the pH to 4.5. They were thenbrought up to 1 ml with 0.25 M RPC-5 buffer (10 mM MgCl,, 10 mMsodium acetate at pH 4.5, and 0.25 M NaCl; molarity refers to NaCl;

MgCl, and sodium acetate remain constant at 10 mM). An equalvolume of phenol saturated with 0.25 M RPC-5 buffer was added toeach and they were blended on a Vortex mixer, combined, andcentrifuged at 27,000 x g for 10 min. Th e aqueous phase was with-drawn and extracted two or three time s with ether saturated with0.25 M RPC-5 buffer. The resulting aqueous phase was gentlywarmed and blended on a Vortex mixer to remove the ether, and thesolution was then either used immediately for chromatography onthe RPC-5 column or frozen for later use. No significant loss oftrichloroacetic acid-precipitable counts or changes in profile wereobserved after storage of aminoacyl-tRNA for as long as 2 weeks.In some cases when the aminoacyl-tRNA was prepared in theabove fashion, spurious peaks were detected which appeared to beassociated with the particular isotope utilized rather than the stage-specific tRNA. In these cases (tyrosine, tryptophan, histidine, andmethioninel, the aminoacyl-tRNA was purified by passing the com-bined reaction mixtures (which has been adjusted to pH 4.5 withacetic acid and mixed with an excess of 0.25 M RPC-5 buffer) over aDEAE -cellulose column poured in a Pasteur pipette. The column hadbeen previously equilibrated with 0.25 M RPC-5 buffer and, followingapplication of the sample, was washed with 20 ml of the same buffer.The tRNA was eluted with 4 ml of 1.0 M RPC-5 buffer, precipitatedwith 2.5 volumes of ice-cold 95% ethanol, and stored at -20 for atleast 15 min. The resulting precipitate was centrifuged at 27,000 x gfor 10 min, drained of ethanol, dried under a stream of air, andresuspended in 0.25 M RPC-5 buffer. Although the DEAE -celluloseisolation method took slightly longer, it yielded much purer prepara-tions of aminoacyl-tRNA and was adopted as the preferred methodfor all isolations in the later stages of this work.

Preparation ofRPC-5 Columns -Materials, preparation and run-ning procedures were as described by Kelmers and Heatherly (281,except as indicated below. RPC-5 absorbent (Miles Laboratories) wassifted through a 200-mesh brass screen and suspended in 0.25 M RPC-5 buffer. The slurry was poured into the column and packed at a flowrate of 3.3 mlimin at a pressure of 250 p.s.i. S everal hundred millili-ters of 0.25 M RPC-5 buffer were passed through the column at thesame flow rate and pressure, followed by several hundred millilite rsof 1.5 M RPC-5 buffer. The column w as then re-equilibrated with 50ml of 0.25 M RPC-5 buffer. A mock run was made using unlabeledcrude ye ast tRN A (Sigma) with a 0.25 to 1.0 M RPC-5 buffer gradient(200 ml) (Pharma cia Fine Che mica ls, Gradient Mixer GM-l). Thecolumn was washed with 50 ml of 1.5 M RPC-5 buffer and re-equilibrated with 0.25 M RPC-5 buffer. Salt concentrations weremonitored with a Radiometer conductivity meter. The initial largewash volumes and the mock run were employed to avoid poor resolu-tion in the first run, a phenomenon which appears to occasionallyoccur with these columns (291.

Analysis and Plottin g of Data -One-milliliter fractions weremixed with 3 ml of Aquasol (New England Nuclear) and counted in arefrigerated scintilla tion counter (Beckman or Searlel. Under theseconditions, the samples formed clear gels which gave constantquench and cross-over throughout the entire range of salt concentra-tions in which the tRNAs eluted. Corrections for background andcross-over were made by a compu ter program (301 which was modi-fied to accept scintillatio n counter data on paper tape for analysis ina Data General Corp. computer. The modified program calculatedthe per cent of the total recovered counts per min for each label ineach fraction (per cent total counts per min), calculated the ratio ofthe per cent total vegetative counts per min to the per cent totaldevelopmental counts per min for each fraction, and plotted this datawith a Hewlett/Packard 7200A graphic plotter. The data for vegeta-tive tRNA was plotted as a single line connecting all data points,that for developmental tRNA was plotted as a dashed line connectingevery other data point, and ratios were plotted as individual points.Salt gradients were drawn in by hand.Sources of Radioactive Amino Acids -Radioactive amino acidswere obtained as follows (specific activities in Curiesimmol): fromNew England Nuclear, L-14C-labeled amino acid mixture, ~-13.Hlarginine (24.21, L-[Clasparagine (0.1791, L-[2,3-:Hlaspartic acid(23.66 and 261, L-13.Hlglutamic acid (20.41, 12-:Hlglycine (10.21, L-1:Hlhistidine hydrochloride (3.131, L-13-:Hlhistidine (10.21, L-14,5-:$HJisoleu cine (105), L-14,5-:Hlleucine (5 and 42.71, L-14,5-z1HJlysine(201, L-[methyl-Hlmethionine (0.1321, L-1:Slmethionine (1001, L-[3-Hlphenylalanine (12.81, L-l3,4-:Hlproline (30 and 381, L-l:Hlserine(1.23 and 3.381, L-l:$Hlthreo nine (2.09 and 2.381, L-1HJtryptophan(1.15 and 7.71, L-13-Cltryptophan (0.05221, L-[3,5-:Hltyrosine (48.2

* A. A. Rizzino, personal commu nication.



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tRNAs of Dictyostelium discoideum 697and 60.3), and L-[3H]valine (1.11); from Ame rshamiS earle, L-[methyl-SH]m ethion ine (8.2); and from SchwarziMann, L-[2,3-1Hla sparagine(12) and all remaining lC-aminoacids (0.050). Unless otherwiseindicated, these amino acids were either generally or uniformlylabeled.

RESULTSOptimal Conditions for Amino Acid Acceptance of Vegeta-tive and Developmental tRNA -Optimal conditions for amino

acid acceptance of vegetative tRNA are described in Table I.All the reaction conditions described gave amino acid incorpo-ration directly proportional to tRNA con centration up to atleast 2.824,,,, units/50 ~1 of reaction mixture, the highest usedfor any of the aminoacylation reactions reported here. Also, allreached and maintained a reproducible plateau value for atleast a 70-min reaction time (Fig. 1). The same conditions wereused with developmental synthetase to determine whether ityielded linear incorporation with vegetative tRNA concentra-tion. For all amino acids tested, this was the case.

We have not rigorously examined the vegetative and devel-opmental aminoacyl-tRNA synthetases for differences in theirability to aminoacylate tRNA. However, in analyses of thechromatographic behavior of the aminoacylated tRNAs wehave found a given tRNA to give similar extents of amino-acylation and an identical profile regardless of whether it isaminoacylated with vegetative or developmental enzymes.Hence the enzymes from one stage are capable of aminoacylat-ing all the isoaccepting species of tRNA from the other stage.Differences in rates of aminoacylation, however, were notinvestigated.

Comparison of Vegetative and Developmental tRNA Prepa-rations for Acceptance of Amino Acids -Using the conditionslisted in Table I, samples of vegetative and developmentaltRNA were aminoacylated with each of the 17 C-aminoacids(Table II). The overall acceptance of the amino acids by thevegetative preparation (1026 pmo l/A,,, unit) is greater thanthat for the developmental one (781 pmol/A,,, unit), indicating

FABLE 1Optimal conditions for amino acid acceptance of vegetative tRNA

Reaction!a en- time atAmm o acid ATP M&l, Amino zyme/lOO whichaad pl assay plateaumix valuereached

VlM P-M nLnArginine 10 20 20 500 20Asparagine 5 10 20 250 20Aspartic acid 5 10 10 500 10Glutamic acid 5 6 60 500 25Glycine 5 10 20 50 20Histidine 5 40 70 500 20Isoleucine 0.625 10 30 500 30Leucine 5 10 20 250 15Lysine 2.5 5 50 100 10Methionine 2.5 5 50 500 50Phenylalanine 5 10 20 50 25Proline 5 10 70 250 20Serine 10 20 40 500 10Threonine 10 10 60 250 25Tryptophan 2.5 5 60 500 30Tyrosine 5 10 20 500 20Valine 5 5 40 100 15

These concentrations var, with the activity of a given synthe-tase preparation and are listed only for purposes of general compari-son.

that in this case the latter preparation is less pure (there wasno systematic difference in purities between vegetative anddevelopmental tRNA preparations). The third column of data,the adjusted ratio of acceptance by the two tRNA prepara-tions, has been corrected for this difference in purity. This was

TIME (midFIG. 1. Aminoacylation of tRNA,. Vegetative tRNA was amino-acylated with IHlleucine and vegetative synthetase according to theassay conditions described in Table I. Four independent experiments

were done, each on a different day. For three the specif ic activity ofthe [Hlleucine was 200 mCiimmo1 and 2.82 A,,, un its of tRNA wereused (A, a, 0); for the other the specif ic activity of the [Hlleucinewas 975 mCiimmo1 and 1.41 A,,, units of tRNA were used (0).

TABLE IIAcceptance of amino acids by vegetative and developmental tRNA

preparationsAminoacylation reactions were run using the conditions noted in

Table I using *C-aminoacids. Counts per min incorporated wereadjusted for counting efficiency (85 to 90%) and the specif ic activity ofthe amino acid and the data expressed as picomole s of amino acidincorporated/A,,,, unit of tRNA. The adjusted ratio of aminoacyla-tion of the vegetative tRNA (V) relative to that of the developmentaltRNA (D) was calc ulate d as I(V)/(D)1 x 1(781)/(1026)]. The quotien t(781)/(1026) corrects for the differenc e in purity between the twopreparations; it is the sum of the picomole s of all amino acidsaccepted by the developmental preparation divided by the corre-_sponding sum for the vegetative preparation.

Picomoles arnm~ acidaccepted/A,,, unit ofAmmo acid VegetativetRNA

D;;;toa;-tRNA

Arginine 60 43Asparagine 51 39Aspartic acid 38 27Glutamic acid 67 47Glycine 72 57Histidine 40 32Isoleucine 94 68Leucine 90 78Lysine 57 37Methionine 10 9Phenylalanine 47 37Proline 43 32Serine 127 97Threonine 95 76Tryptophan 24 20Tyrosine 37 26Valine 74 56Total 1026 781

Adjusted ra-tio of accept-ance

1.061.001.071.090.960.951.050.881.170.850.971.021.000.910.911.081.01



6/11

698 tRNAs of Dictyostelium discoideumdone by multiplying the ratio of the raw data (first columnvalue/second column value) by the ratio for total acceptance(78111026). These adjusted ratios show that within an error ofabout 15% there are no differences in amino acid acceptance bythese preparations.

RPC-5 Profiles of Aminoacyl-tRNAs from Growth and De-velopment - Developmental tRNA, aminoacylated with :H-aminoacid using developmental synthetase, and vegetativetRNA, aminoacylated with C-aminoacid using vegetativesynthetase, were initially co-chromatographed with a steepgradient of 0.25 to 1.0 M RPC-5 buffer (100 ml). This initial runserved two purposes: (a) it indicated the salt concentrationelution range of the aminoacyl-tRNAs and (6) it increased thepeak heights of small peaks, making them easier to detect.Independently isolated tRNA preparations were then amino-acylated, but this time the IX-aminoacid and vegetative syn-thetase were used with developmental tRNA and the H-aminoacid and developmental synthetase were used with thevegetative tRNA. These aminoacyl-tRNA preparations werecombined and run with a very shallow gradient using thepreviously determined salt concentration elution range. Ifthere were any inconsiste ncies between these two runs or ifany aspects of the profiles remained to be clarified, furthercombinations of label and synthetase were used with differenttRNA preparations . In all, up to five vegetative and threedevelopmental tRNA preparations were used for a particularaminoacyl-tRNA. Recovery of labeled material from the col-umns always exceeded 90%.

Figs. 2 to 18 are representative profiles of aminoacyl-tRNAsfrom growth and development. Reproducible quantitative dif-ferences, marked with a K on the figures, are found for someamino acids. The most dramatic quantitative differences arefound in the profile s for asparaginyl-tRN A (Fig. 31, aspartyl-tRNA (Fig. 4), and tyrosyl-tRNA (Fig. 17). The only otherreproducible quantitative differences are in minor peaks: aminor peak for lysine (Fig. lo), the final small peak for methi-onine (Fig. ll), the fourth proline peak (Fig. 131, three minorpeaks for serine (Fig. 14), the minor peak for threonine (Fig.

15), the third peak for tryptophan (Fig. 161, and a minor peakfor valine (Fig. 18 ).

More common than major quantitative differences is thedisplacement of peaks from one stage with respect to the other.In some case s, there is an actual change in the position of thepeak fraction, while in others the displacement is indicatedonly by a change in the ratio of vegetative to developmentalaminoacyl-tRNA indicating changes in the relative amountsof vegetative and developmental tRNA across the peak. Exam-ples of this kind of change in which the developmental peakelutes early (marked with a D on the figures) include themajor arginine peak (Fig. 21, the second aspartic acid peak(Fig. 4), the two major glycine peaks (Fig. 61, the histidinepeak (Fig. 71, the major and two minor isoleucine peaks (Fig.81, the major leucine peak (Fig. 91, the second methionine peak(Fig. ll), the minor pheny lalanine peak (Fig. 121, the twomajor threo nine peaks (Fig. 15), and the major valine peak(Fig. 18). The only example of a displacement in which thevegetative peak elute s early (marked with a V on the figure) isthe second lysine peak (Fig. 10). The displacement in thesecond tyrosine peak (Fig. 17) was not reproduced in all runsand is not marked on the figure.

Reproducibility of RPC-5 Chromatographic Profiles -Theperformance of the column was periodically monitored bypassing Escherichia coli K-12 tRNA (SchwarziMann), amino-acylated with [Hlleucine, through the column, and compar-ing the elution profile with published results (28). A typicalprofile for E. coli leucyl-tRNA is shown in Fig. 19. Columnswere repacked when resoluti on began to deteriorate (usuallyafter about 25 column runs).

RPC-5 chromatographic profiles of Dictyostelium tRNA aresummarized in Table III. Only those profiles which showedsufficient resolution are included in the table. A s indicated inthe table, profiles were generally highly reproducible. Al-though individual tRNA preparations gave consistent profiles,however, some differences between tRNA preparations fromthe same stage were occasionally observed. For example, thequantitative differences in the two major glycine peaks (Fig.

ARGININE4

2io 360 3boFRACTIONRACTION FRACTION

FIG. 2 (lefi). RPC-5 chrom atographic profile of arginyl-tRNA from the column were 204,000 cpm of developmental LHltRNA andfrom vegetative (ueg) and developin g (deu) cel ls. Recovered from the 15,000 cpm of vegetative [Y!ltRNA .column were 108,000 cpm of developmental [H]tRNA and 18,000 cpm FIG. 4 (right). RPC-5 chroma tographic profile of aspartyl-tRNAof vegetative [CltRNA . from vegetative (ueg) and developin g (deu) cel ls. Recovered from theFIG. 3 (center). RPC-5 chromatographic profile of asparaginyl- column were 169,000 cpm of vegetative [HltRNA and 64,000 cpm of

tRNA from vegetative (ueg) and developing (deu) cells. Recovered developmental [WltRNA.



7/11

tRNA.s of Dictyostelium discoideum 699

12 GLUTAMIC ACID

FRACTION FR AC TIO N

L\.160 160 200 220

FRACTIONFIG . 5 (left). RPC-5 chromatographic profile of glutamyl-tRNA column were 221,000 cpm of developmental l:HltRNA and 42,000 cpmfrom vegetative (ueg) and developin g (dew) cel ls. Recovered from the of vegetative [CltRN A.column were 226,000 cpm ofdevelopmental IHJtRNA and 59,000 cpm FIG. 7 (right). RPC-5 chrom atographic profile of histidyl-tR NAof vegetative [ C]tRNA. from vegetative (ueg) and developin g (deu) cel ls. Recovered from theFIG. 6 (center). RPC-5 chroma tographic profile of glycyl-tRNA column were 302,000 cpm of developmental [:HJtRNA and 22,000 cpmfrom vegetative (ueg) and develop ing (deu) cel ls. Recovered from the of vegetative [CltRN A.

ISOLEUCINEIV.0 1 n7% Tj.$06

FRACTION

LEUCINE 4 I j ! L YSIN E A

j5 sclFRAi;ON

Ii0

FIG. 8 (left). RPC-5 chromatographic profile of isoleucyl-tRNA column were 80,000 cpm of developmental lHltRNA and 56,000 cpmfrom vegetative (ueg) and develop ing (deu) cel ls. Recovered from the of vegetative LITltRNA.column were 427,000 cpm ofdevelopmental IHltRNA and 76,000 cpm FIG . 10 (rLgght). RPC-5 chromatograph ic profile of lysyl-tRNAof vegetative I CltRN A. from vegetative (ueg) and develop ing (deal cel ls. Recovered from theFIG . 9 (center). RPC-5 chroma tographic profile of leucyl-tRN A column were 94,000 cpm of vegetative [HltRNA and 33,000 cpm offrom vegetative (ueg) and develop ing (deu) cel ls. Recovered from the developmental L1CltRNA.

6) and the second methionine peak (Fig. 11) were not found inother runs. Also, although quantitative differences in the firstmeth ionine peak (Fig. 11) were reproduced in all runs, whentwo vegetative methionyl-tR NA preparations were co-chro-matographed, they showed quantitative differences of similarmagnitude. Because of results such as these, quantitativedifferences of less than 20%, especially in minor peaks, wereextremely difficult to substantiate. Although such differencesmight exist in viuo, they were beyond the resolution of thisanalysis. In addition, some minor peaks were encounteredin which the peak tube represented less than 0.1% of the total

recovered counts. Peaks in this size range could not be accu-rately analyzed.

It is impos sible for us to equate the number of peaks foundfor a given aminoacyl-tRNA with its number of isoacceptin gspecies . Bes ides the possibility that some peaks are differentmodificatio ns of the same gene product, some may also be dueto the presence of aggregates, tRNA fragments, or amino acidacceptors other than tRN As (15). Since the purpose of thisanalysis was only to screen for differences between vegetativeand developmental tRNA, these other possib ilities have not asyet been pursued.



8/11

700 tRNAs of Dictyostelium discoideum3

METHIONINE 4

FRACTIONFIG. 11 (left). RPC-5 chromatograp hic profile of methiony l-tRNA from the column were 357,000 cpm of developmental [HltRNA and

from vegetative (ueg) and developing (den) cells. Recovered from the 66,000 cpm of vegetative [TltRNA.column were 113,000 cpm of developmental L:!H]tRNA and 46,000 cpm FIG . 13 (right). RPC-5 chroma tographic profile of prolyl-tRNAof vegetative [YS]tRNA. from vegetative (ueg) and develop ing (deu) cel ls. Recovered from theFIG. 12 (center). RPC-5 chromatographic profile of phenylalanyl- column were 125,000 cpm of developmental L3HltRNA and 4000 cpmtRNA from vegetative (ueg) and develop ing (deu) cel ls. Recovered of vegetative [T]tR NA.

I2 SERINE

100 150 200 250 300FRACTION

SO THREONINE4 3 c4

7b 90 II'0FRACTION

1

F2 TRYPTOPHAN,

-jOS

; 0.60d

FRACTIONFIG . 14 (left). RPC-5 chromatographic profile of seryl-tRNA from from the column were 212,000 cpm of vegetative LRHltRNA andvegetative (ueg) and developing (deu) cells. Recovered from the 24,000 cpm of developmental [T]tRNA.colum n were 184,000 cpm of vegetative [H]tRNA and 24,000 cpm of FIG. 16 (right). RPC-5 chromatograp hic profile of tryptophanyl-

developmental [CltRNA. tRNA from vegetative (ueg) and developing (deu) cells. RecoveredFIG. 15 (center). RPC-5 chromatographic profile of threonyl- from the column were 390,000 cpm of vegetative 13HltRNA andtRNA from vegetative (ueg) and develop ing (deu) cel ls. Recovered 19,000 cpm of developmental [TltRNA.DISCUSSION populations parallels the change in amino acid composition of

Comparison of Vegetative and Developmental tRNA for the proteins being made. Hence the concentrations of theAcceptance of Amino Acids -We have found no major differ- tRNAs are believed to adjust to permit efficient synthesis ofence between vegetative and developmental tRNAs in their the specialized proteins (4-6, 31). Since D. discoideum syn-level of aminoacylation with each of the 17 amino acids inves- thesizes many different proteins throughout development, it istigated (Table II). In contrast, significant changes in tRNA not surprising that no major readjustment in levels of aminopopulations do occur in several other developing systems, acid acceptance occur. However, these results do not precludeincluding the silk gland, lens, and reticulocyte (4, 5). All of there being important developmental changes in the tRNAs;these system s, however, are characterized by differen tiation to there could be qualitative changes in the tRNA molecule s, orproduce large quantities of a few proteins having an unusual there could be compensating quantitative changes within theamino acid content, and in each case the change in tRNA subpopulation of isoaccepting specie s for a given amino acid.



9/11

tRNAs of Dictyoste lium discoideum 701

.L.

TYROSINE

IK

245 270 295 320 345FRACTION

FIG. 17 (left). RPC-5 chroma tographic profile of tyrosyl-tRNAfrom vegetative (ueg) and developin g (deu) cel ls. Recovered from thecolumn were 218,000 cpm of developmental 1:HltRNA and 26,000 cpmof vegetative 1CltRNA.

1

TI0.0 L

r0.7 I-2w062

5I-0.5: 0-J

FRACTIONFIG. 19. RPC-5 chromatographic profile of [:HJleucyl-tRNA from

Escherichia coli K-12. A gradient of 0.45 to 1.0 M RPC-5 buffer (200ml) was used. Recovered from the column were 186,000 cpm.

The results of analysis of the tRNAs on reversed phase col-umns, discussed below, indicate changes do occur.

Quantitative Differences between Aminoacyl-tRNAs fromGrowth and 18 H of Development-Quantitative differencesbetween aminoacyl-tRNAs eluted from RPC-5 columns arefound in both major and minor peaks. The most dramaticquantitative differences are found in the profiles for only threeamino acyl-tRNA s: asparaginyl-tRN A (Fig. 31, aspartyl-tRNA(Fig. 4), and tyrosyl-tRNA (Fig. 17). Curiously, these tRNA s,along with histidyl-tRNAs, are the only ones for which m ajor

K; I I,_____ 31 150 a0 110 140 170

FRACTION

VALI NE I

FIG. 18 (right). RPC-5 chroma tographic profile of valyl-tRNAfrom vegetative (ueg) and developin g (deu) cel ls. Recovered from thecolumn were 317,000 cpm of developmental 1:HltRNA and 19,000 cpmof vegetative 1CltRNA .quantitative differences were observed during Drosophilia de-velopment. (32). Since it was shown in that system that differ-ences in a Q base modification were responsible for the shiftingof the aminoacyl-tRNAs from one peak to another, it is possi-ble that a similar post-transcriptional change, rather thanquantitative differences in different gene products, may beresponsible for the analogous differences which we observe.Some of the differences in minor peaks may be due to thepresence of mitochondrial tRNAs. In general, however, differ-ences in minor peaks accounted for a very s mall number of theactual changes observed.

The quantitative difference in the final small peak of methi-onine (Fig. 11) is interesting in light of the restriction inprotein synthesis which occurs early in Dictyostelium develop-ment (33, 34). We do not as yet know when this quantitativechange takes place or if the peak represents a minor cytoplas-mic initiator tRNA.

Displacement of Peaks of Aminoacyl-tRNAs -The mostcommon class of differences between aminoacyl-tRNAs fromgrowth and development is the displacemen t of peaks from onestage with respect to the other. This class of differences ishighly reproducible for all independent tRNA preparationsstudied and does not depend on either the label utilized or thesource of the synthetase in the aminoacylation reaction. Thistype of change is apparent for peaks corresponding to 11 out ofthe 17 amino acids studied. In many cases, the displacemen t isin the major peak, and in all but one case, the developmentaltRNA elutes ahead of the vegetative tRNA. In most instances,a single peak for each amino acid is displaced. Independentconfirmation of peak displaceme nts for three amino acid peakshas been obtained on RPC-3 columns .:

Preferential degradation is not a likely cause for the peakdisplacem ents observed. If there were degradation, it wouldhave to reside in the tRNA preparations themselves sincedisplaceme nts did not change when stage-specific synthetaseswere interchanged in the aminoacylation reactions for theRPC-5 column runs. Such preferential degradation of the

3 M. Brenner, unpublished observations.



10/11

702 tRNAs of Dictyostelium discoideumTABLE III

Summary of RPC-5 chromatographic profilesAminoAcid No. of tRNA prepa-rations analyzed

V% De"No. ofcolumns % Total in Given Peak zt S.D.aT"llS 1 2 3 4 5 6 7-

V 68.3 + 4.4D 71.1 i 1.7R 0.96 z 0.06

31.7 + 4.428.9 T 1.71.11 F 0.1530.6 + 5.151.8 7 1.40.59 7 0.11

A%

ASP

Glu

GU Y

HisIle

LL?U

LY S

Met

Phe

SeII

Thr

TO P

TY ~

Val

4

3

3

2

1

43

3

4

5

3

2

2

2

3

5

2

3

2

2

2

2

33

3

3

3

3

1

2

2

3

3

2

83

3

2

2

66

12

4

8

3

2

2

2

4

6

2

v 2.4 + 0.4D 7.0 + 1.1R 0.35 T 0.03V 65.0 + 3.0D 93.0 7 3.8R 0.70 5 0.03

67.0 + 5.441.2 7 0.41.63 z 0.15

35.0 + 3.07.0 7 3.810.36 T 6.55-

9.1 + 4.2 3.1 + 0.4D 12.2 ? 1.2 3.5 + 1.4R 0.73 i 0.28 0.99 T 0.30- -87.9 + 4.684.4 i 2.51.04 T 0.02-

V 29.7 + 0.5D 28.6 + 9.0R 1.15 + 0.35(one peak only)v 3.0 + 0.6D 2.8 T 0.9R 1.35 3 0.39v 14.9 + 1.3D 15.9 7 1.2R 1.04 z 0.03

70.3 + 0.571.4 ? 9.01.00 T 0.12

8.5 + 0.5 78.0 + 0.712.7 T 1.5 75.5 7 1.40.70 T 0.12 1.03 ? 0.01- -

3.2 + 0.4 5.3 + 0.3 2.1 + 0.12.6 + 0.1 4.6 + 0.3 2.4 ? 0.11.25 7 0.15 1.18 T 0.12 0.91- z 0.09

18.2 + 3.1 6.5 + 0.8 5.7 + 0.618.2 + 4.1 5.8 T 1.0 4.7 7 0.41.03 7 0.08 1.14 T 0.13 1.10 + 0.15- -

53.4 + 2.0 8.9+

+ 1.654.7 2.6 6.0 + 0.70.99 T 0.03 1.04 7 0.08- -

V 10.0 + 5.6D 8.4 + 4.4R 1.16 + 0.12-27.1 + 1.328.3 7 1.70.96 z 0.02

9.6 + 2.614.1 T 1.50.66 T 0.12-

55.7 + 4.552.7 T 2.61.05 IO.05

v 4.2 + 0.2D 6.7 + 1.0R 0.76 T 0.10V 83.3 + 3.0D 87.2 + 1.0R 0.96 + 0.02

33.8 + 4.528.1 + 1.31.22 3 0.2116.7 + 3.012.8 + 1.01.28 E 0.15

58.8 + 4.0 3.3i

+ 0.664.9 1.5 0.3 + 0.10.87 + 0.08 8.17 + 1.42- -

v 11.3 + 5.4D 11.3 + 5.4R 1.19 T 0.18-v 1.3 + 0.7D 0.4 T 0.2R 5.55 + 4.55-v 2.7 + 0.6D 5.0 i 1.3R 0.54 z 0.03V 23.1 +10.6D 24.2 T 7.4R 0.97 ? 0.18-

71.8 +13.574.9 Tl4.40.96 x 0.0187.3 + 2.293.5 i 0.30.93 T 0.02-32.8 + 3.336.1 7 2.60.91 + 0.03-72.3 +11.960.8 T 8.61.13 + 0.06-33.1 + 2.018.4 7 1.31.80 T 0.17-95.3 + 1.896.3 + 2.90.99 IO.01

9.2 + 5.1 7.9 + 3.18.8 T 5.2 5.9 i 3.21.08 T 0.07 1.50 T 0.29- -5.4 + 0.71.9 T 0.12.89 ? 0.54-

64.9 + 3.658.9 7 3.81.10 i 0.014.6 + 1.415.0 T 1.2

0.30 T 0.07-17.5 + 1.6

3.2 F 0.55.53% 1.223.4 + 1.64.3 + 1.7

0.75 z 0.07

1.4 + 0.3 1.0 + 0.2 2.7 + 0.7 1.3 + 0.30.7 + 0.3 0.3 T 0.0 2.5 i 0.9 0.7 T 0.02.07 T 0.37 3.08 T 0.93 1.12 T 0.12 1.82 0.38- - z

V 41.6 + 2.8D 77.1 7 2.1R 0.57 i 0.03

5.6 + 0.4 3.7 + 1.4l.lT 0.4 0.7 7 0.37.16 z 2.34 5.20 i 0.64-

v 2.3 + 0.7D 0.8 + 0.2R 2.95 ? 0.28

aThe first lne for each amino acid (V) gives the percentages of the total cpm of vegetative aminoacyl-tRNA found in each peak,and the second lne (D) gives the corresponding values for developmental tRNA. The third lne (R) gives the ratio for each peakof the percentage vegetative tRNA to the percent developmental tRNA. All data are the averages for the number of columns indicated,plus or minus the standard deviation of the mea n. (Note that the third lne is not the quotien t of the firs t lne divided bythe second, that being the ratio of the averages rather than the average of the ratios.)

tRNA preparations is unlikely because (a) vegetative and dence of degradation, (d) rates and extents of aminoacylationdevelopmental tRNAs were prepared in identical fashion, (b) were identical for tRNAs from both stages, even when stage-displacem ents were consistent for five different vegetative and specifi c synthetases were switched, suggesting (but not prov-three different developmental tRNA preparations, (c) gel elec- ing) that synthetase recognition was identical for both, (e)trophoretic analyses of RNA preparations showed little evi- only certain peaks w ere affected, and (f) heatin g of vegetative



11/11

tRNAs of Dictyostelium discoideum 703tRNA, to 80 for 10 min (35) did not cause any peaks to be Acknowledgments -We are extremely grateful to Paula A.displaced. We cannot, of course, completely rule out the possi- Palatnik for help with the computer analysis of the data and tobility of preferential degradation without doing fingerprint Larry E. Field s and Macy Koehle r for help with the experi-analyses. mental work.

Nature ofDifferences in Aminoacyl-tRNAs between Growthand Development-Although some of the peak displaceme ntsmay result from quantitative differences within a heteroge-nous peak, others are probably due to differences in post-transcriptional modification between vegetative and develop-mental tRNA. Levels of acceptance of aminoacyl-tRNAs fromboth stages are extremely similar. In addition, in most of thecases in which a peak displacemen t is observed, the overallprofile and the heights of the specif ic peaks in question arealso strikingly similar. Even the differences observed in theaspartic acid, asparagine, and tyrosine profiles may representdifferences in modification, as was demonstrated for changesin these same tRNAs during Drosophilia development (32).The fact that displaced peaks alm ost always have the develop-mental tRNA eluting ahead of the vegetative tRNA furthersuggests that a common change or a very small number ofchanges in modification of these tRNAs are occurring duringDictyostelium development. In the his T mutants of Salmo-nella, for example, it was shown that a lack of pseudouridinein the anticodon loop, caused by the absence of a single modify-ing enzyme activity, resulted in at least 10 peak displace-ments, corresponding to several different amino acids. Allpeak displacem ents were in the same direction (8, 9, 36).

REFERENCES1234.5.67.8.

Sueoka. N.. and Kane-Sueoka. T. (1970)Proe. Nucleic Acid Res.Mol. siot. 10, 23-55 Littauer. U. Z.. and Inouve. H. (1973) Annu. Reu. Biochem. 42,439-470

Scherberg, N. H., and Weiss , S. R. (1972) Proc. Natl. Acad. Sci.U. S. A. 69, 1114-1118Garel, J-P. (1974) J. Theor. Biol. 43, 211-225Smith, D. W. E. (1975) Science 190, 529-535Garel, J-P. (1976) Nature 260, 805-806Ilan. J.. and Ilan. J. (19751 Curr. T O D . Deu. Biol. 9. 88-136Cortese; R., Landsbe rg, R., Vender Haar, R. A., Umbarger, H.E., and Ames, B. N. (1974) Proc. Natl. Acad. Sci. U. S. A. 71,1857-1861

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Rizzino, A. A., Bresa lier, R. S., and Freund lich, M. (1974) J.Bacterial. 17. 449-455Lodish, H. F., Jacobson, A., Firtel, R., Alton, T., and Tuchman,J. (19741 Proc. Natl. Acad. Sci. U. S. A. 71, 5103-5108Jacobson, A., Firtel, R. A., and Lodish, H. F. (197415. Mol. Biot.82, 213-230Brenner, M., Tisdale , D., and Loomis, W. F., Jr. (1975)Exp. CellRes. 90, 249-252Loomis. W. F.. Jr. (1975) Dictvostelium discoideum : A Devetoo-mental System, Academ ic Press, New YorkBrenner. M.. and Ames. B. N. (1971) in Metabolic Pathways(Vogel, H.J., edj Vol. 5, pp. 349-387, Academ ic Press, NewYork

Peak Displaceme nts Are Not Unique to Dictyostelium De-velopment -The observation that there were peak displace-ments for most of the amino acids tested prompted us to re-examine published profiles of aminoacyl-tRNAs in other de-veloping systems to see if peak displacements could also bedetected. Such examination suggests that displacem ents arepresent. Some examples include the lysyl- and methionyl-tRNA s of developin g brine shrimp (Ref. 37, p. 30), the seryl-tRNA of milkweed bugs undergoing embryogenesis (Ref. 38, p.127), and the histidyl-tRNA from different tissues duringmouse development (Ref. 39, pp. 68-69). Peak displaceme ntsare therefore probably not unique to Dictyo stelium develop-ment and should be considered as examples of unresolvedheterogeneity, possibly due to differences in post-transcrip-tional modification.

15.16.17.18.19.20.21.22.23.24.25.26.27.28.29.30.31.32.33.34.

Soffer, R. L. (1974)Adu. Enzymol. Relat. Areas Mo l. Biol. 40,91-139Marin. F. T. (1976) Dev. Biol. 48. 110-117Loomis, W. F., Jr. (1971) Exp. C ell Res. 64, 484-486Coccuci. S.. and Sussman M. (1970) J. Cel Biol. 45, 399-407Watts, D. J., and Ashworth, J. M. (1970) Biochem. J. 119, 171-174Brenner, M., and Am es, B. N. (1972) J. Biol. Chem. 247, 1080-1088Sarin, P. S., and Zamecnik, P. C. (1964) Biochim. Biophys. Acta

91, 653-655

Relationship of Differences to Translational Control -Be-cause of the central position occupied by tRNAs in proteinsynthesis, any analysis of tRNA chang es during developmentmust consider the postulated role of tRNA in translationalcontrol (1). The possibility that some of the differences weobserve may represent differences in Q base modification inthe anticodon loop is of interest in this regard. In preliminarywork with an in vitro protein synthesizing system from wheatgerm programmed with Dictyostelium vegetative mRNA,however, tRNAs from both stages appeared to functionequally well in their ability to stimulate incorporation ofradioactive amino acids into hot trichloroacetic acid-precipita-ble material. This approach is presently being extended bycomparing on gels products synthesized in vitro with stage-specif ic tRNAs.

Layne, E. (1957) Methods Enzymol. 3, 450-451Kissane, J. M., and Robins, E. (1958) J. Biol. Chem . 233,184-188White, B. N., Tener, G. M., Holden, J., and Suzuki, D. T. (1973)

Deu. Biol. 33, 185-195Lowry, 0. H., Rosebroug h, N. J., Farr, A. L., and Rand all, R. J.(1951) J. Biol. Chem. 193, 265-275Rubin, I. B., Mitchell, T. J., and Goldstein, G. (1971) Anal.

Chen. 43, 717-721Bonnett, J., and Ebel, J. (1972) Eur. J. Biochem. 31, 335-344Kelmers, A. D., and Heatherly, D. E. (1971) Anal. Biochem. 44,486-495Roe, B., Marcu, K., and Dudock, B. (1973) Biochim. Biophys.Acta 319, 25-36

Yund, M. A., Yund, E. W., and Kafatos, F. C. (1971)Biochen.Biophys. Res. Comm un. 43, 717-722Delaney, P., and Siddiqui, M. A. Q. (1975) Dew. Biol. 44,54-62

White, B. N., Tener, G. M., Holden, J., and Suzuki, D. T. (1972)J. Mol. Biol. 74, 635-651Tuchman, J., Alton, T., and Lodish, H. F. (1974) Deu. Biol. 40,116-128Lodish, H., Alton, T., Margolskee, J., Dottin, R., and Weiner, A.(1975) in Developmental Biology: Pattern Formation, Gene

Regulation, ICN-UCLA Symposium on Molecular and Cellu-lar Biology (McMahon, D., and Fox, C. F., eds) Vol. 2, pp. 366-383. W. A. Beniamin. Menlo Park, Ca.

The possibility that some of the tRNA changes affect func-tions other than translation should also be considered. Theanalogy of the peak displacem ents observed here to thoseresulting from the his T mutation in Salmone lla, which isknown to affect gene transcription (8, 9, 141, has already beenpointed out.

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isolation and characterization of transfer rnas from dicty during growth and development

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