: Isolation and characterization of transfer RNAs from Dictyostelium discoideum during growth and development.

C M Palatnik and E R Katz

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

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and Characterization of Transfer RNAs from DictyosteZium discoideum during Growth and Development*
(Received CARL MATHEW PALATNIK~ AND EUGENE R. KATZ for publication, August 13, 1976)

Isolation

From the Department Brook, Stony Brook,

MICHAEL BRENNERS:

of Cellular and New York 11794 Laboratories,

Comparative

Biology,

State

University

of New

York

at Stony

From

the Biological

Harvard

University,

Cambridge,

Massachusetts

02138

Transfer RNAs and aminoacyl-tRNA synthetases were isolated from vegetative and developing cells of the cellular slime mold Dictyostelium discoideum. Using the homologous synthetases, the tRNAs were compared for their levels of acceptance of 17 amino acids. These levels were found to be the same. The tRNAs were further compared by double label chromatography on reversed phase columns. Some major quantitative differences in individual peaks were observed. In addition, for a number of aminoacyl-tRNAs, small but reproducible displacements of individual peaks were apparent. In many cases the displacement was in the major peak, and in all but one case the developmental peak eluted ahead of the vegetative peak. Taken together, these results suggest that there is no change from growth to development in the transcription of tRNA genes, but that there are important changes in post-transcriptional modification.

Differences in chromatographic mobilities have been demonstrated for tRNAs from a wide variety of tissues in different metabolic or developmental states (for detailed reviews see Refs. 1 and 2). Since the existence of such differences is quite common, an understanding of their nature and functional significance is of general biological interest. In some highly specialized differentiating systems, tRNAs have been shown to adapt to the amino acid composition of the few major proteins being synthesized (3-61, and therefore appear to be components of the differentiation process itself. In a few of these cases there is also some evidence that the availability of a few tRNAs may limit and thereby regulate the rate of synthesis of these proteins (5, 7). However, most changes in metabolic or developmental state do not involve gross changes in the amino acid composition of the proteins being made. In these more common instances it has proved difficult to proceed beyond observations of correlations between changes in tRNAs and * This work was supported by grants to E. R. K. from the United States Public Health Service (GM 18476) and to M. B. from the Milton Fund, Clark Fund, and the National Science Foundation (GB 32042 and BMS 72.01830 A02). $ Present address, Department of Microbiology, University of Massachusetts Medical School, Worcester, Mass. 01605. 5 To whom correspondence should be addressed.

changes in the biological state of cells to the goal of understanding cause and effect relationships. Only in a few studies with bacteria has it been possible to ascribe functions to the tRNA changes. In Salmonella typhimurium a tRNA modification has been found essential for proper transcriptional control of several amino acid biosynthetic operons (8, 9). For technical reasons, eukaryotes have not been as amenable to analysis as bacteria. Except for the highly specialized systems noted above, the significance of tRNA changes in eukaryotic systems remains unknown. Among eukaryotic organisms, the cellular slime mold Dictyostelium discoideum appears to be especially promising for pursuing an analysis of developmental changes in tRNAs beyond the stage of simple correlation. In addition to its ease of manipulation and the fact that it differentiates in 24 h into only two cell types, the technology exists for assaying for the transcriptional and translational consequences of any detected alterations. Wheat germ and rabbit reticulocyte cell-free protein-synthesizing systems have been shown to be extremely efficient in translating stage-specific Dictyostelium mRNAs (10). Furthermore, an in vitro system for synthesizing mRNA precursor, using isolated nuclei, has been developed and has been shown to give excellent initiation (111. Finally, the haploid nature of the organism facilitates obtaining mutants (12, 13). The utilization of mutants to elucidate the role of tRNA in the repression of certain amino acid biosynthetic operons in bacteria (14) indicates the utility of this feature. We are interested in the possibility that tRNAs might be involved in regulation of either translational or transcriptional events during development of D. discoideum. Because of the well known role of tRNA in decoding the base sequence of mRNA during protein synthesis, considerable attention has been given to the possibility that tRNAs may be involved in regulating the translation of messenger RNAs (1, 2). But in addition to their part in translation, tRNAs act also to donate amino acids to lipids, peptidoglycans, and proteins (15); and in bacterial systems at least, have been implicated in the transcriptional regulation of certain amino acid biosynthetic pathways (8, 9, 14). Hence tRNAs may assume multiple roles within an organism, including functioning as regulatory agents at several levels of control. I C. T. Mabie and A. Jacobson, personal communication.

694

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tRNAs

o/Uictyostelium

discoideum

695

We have become intrigued by the recent finding that starvation for certain amino acids is apparently required for initiating development (16). Changes in in uiuo levels of tRNA aminoacylation could conceivably alter the protein synthetic patterns in early development. In addition, by analogy with the role of tRNAs in expression of certain amino acid biosynthetic operons in bacteria (8, 9, 141, the signal for amino acid starvation and consequently the signal for initiation of development in Dictyostelium may well be communicated to the transcriptional control apparatus by tRNAs. In order to provide a broad framework from which to initiate a functional analysis of tRNAs in Dictyostelium development, a general characterization of tRNAs present during growth and 18 h of development (initiation of culmination) was made. This developmental stage was chosen as it allowed a maximum of time for changes in tRNAs and aminoacyl-tRNA synthetases to accumulate. Still later times were not satisfactory because during culmination stalk cells extrude their contents, vacuolate, and die. Following the isolation of tRNAs and aminoacyl-tRNA synthetases, we have determined the reaction conditions for optimum aminoacylation of the tRNAs with each of 17 amino acids, and this has in turn enabled us to assay for quantitative changes in acceptance for each of these amino acids during development. Finally, we have analyzed isoaccepting species of aminoacyl-tRNAs using reversed phase column chromatography. The combined results of these studies suggest that during Dictyostelium development tRNAs are regulated primarily at the level of post-transcriptional modification.
MATERIALS AND METHODS

Culturing

and

Harvesting

of

Cells-Dictyostelium

discoideum

strain A3 (17) was grown at 22-23 on a gyratory platform shaker rotating at 175 to 200 rpm. Cells were grown in HL-5 medium containing yeast extract (Difco), proteose peptone (Difco), glucose and phosphate buffer at pH 6.5 (18, 19). Cultures containing 2 liters of medium were routinely grown in 4-liter flasks and had a generation time of 9 to 10 h. Exponentially growing cells at concentrations of less than 6 x 10 cells/ml were harvested in a Sorvall RC-2B centrifuge at 1500 x g for 30 s at 4 or in a Sharples continuous flow centrifuge. growing cells were harvested, washed once at a titer of 1 x lO/ml with PDF buffer (40 rnM KCl, 10 rnM MgCl,, 18.5 mM K,HPO,, 26.5 mM KH,PO,, and 500 kg/ml of streptomycin sulfate, pH 6.4 to 6.61, and resuspended in PDF buffer at a final concentration of 3 x 10 cells/ml. Five milliliters of the cell suspension 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 Petri dish. The filters had been previously saturated with 40 ml of PDF buffer and freed of air bubbles with a glass spreader. Petri dishes were placed in polyethylene bags on flat trays containing moist paper towels. Bags were sealed with tape and incubated at 22. After 18 h of development, just before culmination commenced, cells were scraped from the filters and washed once. Details of wash buffers are described in subsequent sections. Only cells with an apparent synchrony of greater than 80% were used. Preparation of tRNA -Transfer RNA was prepared by a modiflcation of a method originally utilized for isolation of tRNA from Salmonella typhimurium (20). Transfer RNAs from exponentially growing cells (vegetative tRNA) and from cells at 18 h of development (developmental tRNA) were isolated in identical fashion. It was essential that freshly harvested cells be used for the tRNA preparations, as frozen 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 resuspended in TMS buffer at a final concentration of 2 ml/g of cells. An equal volume of phenol (Mallinckrodt), saturated with TMS buffer, was added and the mixture was shaken vigorously for a minimum of 1 h at 4. The resulting suspension was centrifuged at 23,000 x g for 30
Plating Cells Cells -Exponentially

for

Development

and

Haruesting

of

Developing

min. The aqueous phase was withdrawn, 1150 volume of 5 M NaCl added to give a final concentration of 0.1 M NaCl and 2.5 volumes of 95% ethanol added. The solution was stored at -20 for up to a month without any deleterious effects, during which time other phenol extracts were prepared and combined with it. As much ethanol as possible was removed with an aspirator and the remaining precipitate collected by centrifugation at 16,000 x g for 30 min. The pellet was dried under vacuum, resuspended in 0.02 M NaCl in TMS buffer, and extracted twice with an equal volume of ether saturated with the same buffer. The resulting solution was passed onto a DEAE-cellulose column (Whatman DE52, l-ml bed volume/g of cells) previously equilibrated with 0.02 M NaCl in TMS buffer. The column was washed at a flow rate of 2 to 5 mlimin until the A,,, of the effluent was 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 buffer at a flow rate of 1 mlimin. All fractions with an A,,, of greater than 1.0 were pooled and the tRNA precipitated by addition of 2.5 volumes of 95% ethanol followed by overnight storage at -20. The precipitate was collected by centrifugation at 16,000 x g for 30 min. The precipitate was dried under vacuum, redissolved in 1.8 M Trisiacetate, pH 8.0, and any attached amino acids removed by incubation at 37 for 90 min (21). Ice-cold 95% ethanol (2.5 volumes) was added and the tRNA precipitate kept at -20 for at least 15 min to ensure complete precipitation. The precipitate was collected by centrifugation at 27,000 x g for 15 min, washed twice with 75% ethanol containing 0.1 M NaCl, dried under vacuum at room temperature, and stored as a powder at -20. The A&APHO of tRNA prepared in this manner was 2 and the average yield was about 1 mgig of cells. The A,,,/mg of tRNA in a buffer of 100 mM sodium cacodylate, pH 7.5, 10 mM MgCl,, and 2 mM Na&O, was 15. The tRNA preparations contained about 5% protein as determined by the biuret method (22) and less than 0.1% primary aldehydes (which include DNA) as measured by the diaminobenzoic acid assay (23). There were no apparent differences in these properties for vegetative and developmental tRNA preparations. Electrophoresis on polyacrylamide gels showed the tRNA preparation to be about 10% pure before the DEAE-cellulose chromatography and 50 to 60% pure at the final step, the primary contaminant being ribosomal RNA. This estimate of purity agrees well with the results from aminoacylation (Table II). Assuming that alanine, glutamine, and cysteine are accepted by the preparations to the same extent as the average of the other 17 amino acids, a total of about 1060 pmol of amino acids were accepted per A,,, unit of tRNA (average for the vegetative and developmental preparations shown in Table II). Since pure tRNAs accept about 1700 pmol of amino acids/A,,,, unit (241, on the basis of amino acid acceptance our preparation was also about 60% pure. Preparation of Aminoacyl-tRNA Synthetases - Aminoacyl-tRNA synthetases from exponentially growing cells (vegetative synthetase) and from cells at 18 h of development (developmental synthetase) were prepared in identical fashion. Cells were harvested, washed once at a titer of 1 x lOHim with cold 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/ml. Cells were homogenized with a tight fitting Dounce homogenizer until 90% breakage was achieved. Lower cell concentrations resulted in less efficient breakage. Alternate procedures for cell breakage (sonication, freezethawing, detergent lysis) gave much poorer activities. The A,,, of the homogenate was adjusted to about 80 and the homogenate centrifuged at 37,000 x g for 30 min at 4. The clear supernatant was removed, transferred to an ultracentrifuge, and centrifuged at 105,000 x g for 1 h. Failure to dilute the cell extract occasionally yielded preparations of reduced activity, probably because particulate material remaining in suspension interfered with the DEAE-cellulose chromatography which followed. The supernatant from the 105,000 x g centrifugation step was withdrawn and l/q volume of 1 M KC1 was added to give a final concentration of 0.2 M KCl. It was then passed onto a DEAE-cellulose column (Whatman DE52, 6-ml bed volume/g of cells), which had been previously equilibrated at 4 with buffer containing 0.2 M KCl, 50 mM TrisiHCl, pH 7.5, 10 mM MgCl,, 10% glycerol, and 14 rnM mercaptoethanol. It was eluted directly with the same buffer. Fractions contammg an ALGO/APHO of less than 1.2 (greater than 95% protein) were pooled. The pooled fractions were concentrated and free amino acids were removed by vacuum dialysis against two changes of synthetase buffer, the second containing 50% glycerol.

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696

tRNAs

of Dictyostelium

discoideum

The first dialysis was for a minimum of 2 h against at least 10 volumes of buffer and the second was for a minimum of 4 h against at least 200 volumes of buffer. Alternatively, the pooled column effluent was brought to 85% saturation with enzyme grade ammonium sulfate (SchwarziMann) and the resulting precipitate was dialyzed against two changes of at least 200 volumes of synthetase buffer for at least 2 h each before being brought up to 50% glycerol. The column step was necessary both to remove tRNA from the enzyme preparation and to eliminate some component(s) which reduced both the rate and extent of the aminoacylation reactions. This component was not removed by either ammonium sulfate precipitation or dialysis. Some preliminary experiments eliminated trivial explanations for the inhibition: it did not result from simple exhaustion of ATP, amino acid, or enzyme, or from complexing of MgCl,. Nor did it appear to be due to degradation of tRNA or accumulation of pyrophosphate or 5AMP. The nature of the inhibition was not further pursued. Synthetase preparations were stored at -20 and were stable for at least 6 months. Final protein concentrations (2.51 were 25 to 30 mgi ml in preparations concentrated with ammonium sulfate and 50 to 65 mgiml in preparations concentrated by vacuum dialysis. Incubations in aminoacylation reactions with the tRNA omitted gave very low background counts, indistinguishable from reaction mixtures lacking both synthetase and tRNA, indicating that synthetase preparations were not contaminated with tRNA. No tRNA degradative properties were detected in synthetase preparations during aminoacylation reactions, as all aminoacylation reactions reached and maintained reliable plateaus which were unaffected by preincubating the tRNAs with synthetases before adding labeled amino acid. Chromatography on reversed phase columns also indicated an absence of degradative activities in the synthetase preparations. The chromatographic profiles showed sharp peaks and were highly reproducible regardless of the synthetase preparation used for the aminoacylation. In addition, vegetative tRNA aminoacylated with vegetative synthetase for 5 min with 1Hlleucine and vegetative tRNA aminoacylated with vegetative synthetase for 60 min with [Clleucine, gave identical profiles when co-chromatographed. Optimization of Assay Conditions - Assays were performed at 22 since this is the temperature for optimal growth ofD. discoideum. (A few assays conducted at 37 gave no activity). The buffer and pH were selected by optimizing for the acceptance of leucine by the tRNA. Cacodylate buffers gave levels of incorporation higher than buffers of phosphate or Tris, and a pH of 7.5 was found superior to 6.5, 7.0, and 8.0. Other reaction components were individually optimized for each of the amino acids, using the following procedure. A standard 50 ~1 of assay mixture contained 100 mM sodium cacodylate, pH 7.5, 5 mM ATP, 10 mM MgCI,, 10 pM IH-aminoacid, 50 FM concentration each of the other 19 unlabeled amino acids (except that asparagine, glutamine, and glutamic acid were omitted from the aspartic acid reaction mixture; glutamine and asparagine from the glutamic acid mixture; and glutamine and glutamic acid from the asparagine mixture) and about 2.5 A,,;,, units of vegetative tRNA. Reactions were terminated by precipitation on filter paper discs with 5% trichloroacetic acid according to the procedure of Rubin et al. (26) and counted in a toluene-based scintillation fluid containing 4 g of 2,5-diphenyloxazole (PPO) and 0.1 g of 1,4-bisl2-(5.phenyloxazolylllbenzene (POPOPJiliter of toluene. The first step in the optimization procedure was to vary the concentration of the vegetative synthetase. The concentration which yielded and maintained the highest plateau level was chosen for subsequent assays. The concentration of the synthetase was one of the most critical variables and had to be carefully determined for each enzyme preparation. Concentrations which were too low reduced both the rate and extent of the reaction, whereas concentrations which were too high did not maintain a plateau value. Similar observations have been made in other systems (27). After the optimum enzyme concentration was obtained, the concentration of the H-aminoacid was varied until the plateau was no longer dependent on the amino acid concentration. Finally, the MgCl, and ATP concentrations were varied both together and independently. Once the reaction components were optimized, the reaction was tested to ascertain if the incorporation was linear with tRNA concentration. Preparation of Aminoacyl-tRNA for Columns -Following aminoacylation of the tRNA from both stages under conditions which gave optimum aminoacylation, the reaction mixes from each stage were made 0.01 M with acetic acid to reduce the pH to 4.5. They were then brought up to 1 ml with 0.25 M RPC-5 buffer (10 mM MgCl,, 10 mM sodium acetate at pH 4.5, and 0.25 M NaCl; molarity refers to NaCl;

MgCl, and sodium acetate remain constant at 10 mM). An equal volume of phenol saturated with 0.25 M RPC-5 buffer was added to each and they were blended on a Vortex mixer, combined, and centrifuged at 27,000 x g for 10 min. The aqueous phase was withdrawn and extracted two or three times with ether saturated with 0.25 M RPC-5 buffer. The resulting aqueous phase was gently warmed and blended on a Vortex mixer to remove the ether, and the solution was then either used immediately for chromatography on the RPC-5 column or frozen for later use. No significant loss of trichloroacetic acid-precipitable counts or changes in profile were observed after storage of aminoacyl-tRNA for as long as 2 weeks. In some cases when the aminoacyl-tRNA was prepared in the above fashion, spurious peaks were detected which appeared to be associated with the particular isotope utilized rather than the stagespecific tRNA. In these cases (tyrosine, tryptophan, histidine, and methioninel, the aminoacyl-tRNA was purified by passing the combined reaction mixtures (which has been adjusted to pH 4.5 with acetic acid and mixed with an excess of 0.25 M RPC-5 buffer) over a DEAE-cellulose column poured in a Pasteur pipette. The column had been previously equilibrated with 0.25 M RPC-5 buffer and, following application 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, precipitated with 2.5 volumes of ice-cold 95% ethanol, and stored at -20 for at least 15 min. The resulting precipitate was centrifuged at 27,000 x g for 10 min, drained of ethanol, dried under a stream of air, and resuspended in 0.25 M RPC-5 buffer. Although the DEAE-cellulose isolation method took slightly longer, it yielded much purer preparations of aminoacyl-tRNA and was adopted as the preferred method for all isolations in the later stages of this work. Preparation ofRPC-5 Columns -Materials, preparation and running procedures were as described by Kelmers and Heatherly (281, except as indicated below. RPC-5 absorbent (Miles Laboratories) was sifted through a 200-mesh brass screen and suspended in 0.25 M RPC5 buffer. The slurry was poured into the column and packed at a flow rate of 3.3 mlimin at a pressure of 250 p.s.i. Several hundred milliliters of 0.25 M RPC-5 buffer were passed through the column at the same flow rate and pressure, followed by several hundred milliliters of 1.5 M RPC-5 buffer. The column was then re-equilibrated with 50 ml of 0.25 M RPC-5 buffer. A mock run was made using unlabeled crude yeast tRNA (Sigma) with a 0.25 to 1.0 M RPC-5 buffer gradient (200 ml) (Pharmacia Fine Chemicals, Gradient Mixer GM-l). The column was washed with 50 ml of 1.5 M RPC-5 buffer and reequilibrated with 0.25 M RPC-5 buffer. Salt concentrations were monitored with a Radiometer conductivity meter. The initial large wash volumes and the mock run were employed to avoid poor resolution in the first run, a phenomenon which appears to occasionally occur with these columns (291. Analysis and Plotting of Data -One-milliliter fractions were mixed with 3 ml of Aquasol (New England Nuclear) and counted in a refrigerated scintillation counter (Beckman or Searlel. Under these conditions, the samples formed clear gels which gave constant quench and cross-over throughout the entire range of salt concentrations in which the tRNAs eluted. Corrections for background and cross-over were made by a computer program (301 which was modified to accept scintillation counter data on paper tape for analysis in a Data General Corp. computer. The modified program calculated the per cent of the total recovered counts per min for each label in each fraction (per cent total counts per min), calculated the ratio of the per cent total vegetative counts per min to the per cent total developmental counts per min for each fraction, and plotted this data with a Hewlett/Packard 7200A graphic plotter. The data for vegetative tRNA was plotted as a single line connecting all data points, that for developmental tRNA was plotted as a dashed line connecting every other data point, and ratios were plotted as individual points. Salt gradients were drawn in by hand. Sources of Radioactive Amino Acids -Radioactive amino acids were obtained as follows (specific activities in Curiesimmol): from New 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, L1:Hlhistidine hydrochloride (3.131, L-13-:Hlhistidine (10.21, L-14,5:$HJisoleucine (105), L-14,5-:Hlleucine (5 and 42.71, L-14,5-z1HJlysine (201, L-[methyl-Hlmethionine (0.1321, L-1:Slmethionine (1001, L-[3Hlphenylalanine (12.81, L-l3,4-:Hlproline (30 and 381, L-l:Hlserine (1.23 and 3.381, L-l:$Hlthreonine (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 communication.

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tRNAs

of

Dictyostelium

discoideum

697

and 60.3), and L-[3H]valine (1.11); from AmershamiSearle, L-[methylSH]methionine (8.2); and from SchwarziMann, L-[2,3-1Hlasparagine (12) and all remaining lC-aminoacids (0.050). Unless otherwise indicated, these amino acids were either generally or uniformly labeled.
RESULTS

that in this case the latter preparation is less pure (there was no systematic difference in purities between vegetative and developmental tRNA preparations). The third column of data, the adjusted ratio of acceptance by the two tRNA preparations, has been corrected for this difference in purity. This was

Optimal Conditions for Amino Acid Acceptance of Vegetative 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 incorporation directly proportional to tRNA concentration up to at least 2.824,,,, units/50 ~1 of reaction mixture, the highest used for any of the aminoacylation reactions reported here. Also, all reached and maintained a reproducible plateau value for at least a 70-min reaction time (Fig. 1). The same conditions were used with developmental synthetase to determine whether it yielded linear incorporation with vegetative tRNA concentration. For all amino acids tested, this was the case. We have not rigorously examined the vegetative and developmental aminoacyl-tRNA synthetases for differences in their ability to aminoacylate tRNA. However, in analyses of the chromatographic behavior of the aminoacylated tRNAs we have found a given tRNA to give similar extents of aminoacylation and an identical profile regardless of whether it is aminoacylated with vegetative or developmental enzymes. Hence the enzymes from one stage are capable of aminoacylating all the isoaccepting species of tRNA from the other stage. Differences in rates of aminoacylation, however, were not investigated. Comparison of Vegetative and Developmental tRNA Preparations for Acceptance of Amino Acids -Using the conditions listed in Table I, samples of vegetative and developmental tRNA were aminoacylated with each of the 17 C-aminoacids (Table II). The overall acceptance of the amino acids by the vegetative preparation (1026 pmol/A,,, unit) is greater than that for the developmental one (781 pmol/A,,, unit), indicating

TIME

(mid

FIG. 1. Aminoacylation of tRNA,. Vegetative tRNA was aminoacylated with IHlleucine and vegetative synthetase according to the assay conditions described in Table I. Four independent experiments were done, each on a different day. For three the specific activity of the [Hlleucine was 200 mCiimmo1 and 2.82 A,,, units of tRNA were used (A, a, 0); for the other the specific activity of the [Hlleucine was 975 mCiimmo1 and 1.41 A,,, units of tRNA were used (0). TABLE Acceptance II

FABLE

Optimal

conditions

for amino acid acceptance

ATP M&l, Amino aad

of vegetative
!a enzyme/lOO pl assay mix

tRNA
Reaction time at which plateau value reached nLn

Ammo

acid

and developmental tRNA preparations Aminoacylation reactions were run using the conditions noted in Table I using *C-aminoacids. Counts per min incorporated were adjusted for counting efficiency (85 to 90%) and the specific activity of the amino acid and the data expressed as picomoles of amino acid incorporated/A,,,, unit of tRNA. The adjusted ratio of aminoacylation of the vegetative tRNA (V) relative to that of the developmental tRNA (D) was calculated as I(V)/(D)1 x 1(781)/(1026)]. The quotient (781)/(1026) corrects for the difference in purity between the two preparations; it is the sum of the picomoles of all amino acids accepted by the developmental preparation divided by the corre_ _ sponding sum for the vegetative preparation. Picomoles arnm~ acid accepted/A,,, unit of Adjusted raAmmo acid

of amino acids by vegetative

VlM

P-M

Arginine Asparagine Aspartic acid Glutamic acid

Glycine

10 5
5

20 10
10

20 20
10

500 250
500

20 20
10

Vegetative tRNA
60 51 38 67 72 40 94 90 57 10 47 43 127 95 24 37 74 1026

D;;;toa;tRNA 43 39 27 47 57 32 68 78 37 9 37 32 97 76 20 26 56 781

tio of acceptance 1.06 1.00 1.07 1.09 0.96 0.95 1.05 0.88 1.17 0.85 0.97 1.02 1.00 0.91 0.91 1.08 1.01

Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

5 5 5 0.625 5
2.5 2.5 5 5

6 10 40
10

10
5 5 10 10 20

60 20 70 30 20
50 50 20 70 40

500 50 500 500 250

100 500 50 250 500 250 500 500 100

25 20 20 30 15
10 50 25 20 10 25 30 20 15

10 10
2.5 5 5

10
5 10 5

60
60 20 40

These concentrations var, tase preparation and are listed son.

with the activity only for purposes

of a given syntheof general compari-

Arginine Asparagine Aspartic acid Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Total

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698

tRNAs

of Dictyostelium

discoideum 15), the third peak for tryptophan (Fig. 161, and a minor peak for valine (Fig. 18). More common than major quantitative differences is the displacement of peaks from one stage with respect to the other. In some cases, there is an actual change in the position of the peak fraction, while in others the displacement is indicated only by a change in the ratio of vegetative to developmental aminoacyl-tRNA indicating changes in the relative amounts of vegetative and developmental tRNA across the peak. Examples of this kind of change in which the developmental peak elutes early (marked with a D on the figures) include the major arginine peak (Fig. 21, the second aspartic acid peak (Fig. 4), the two major glycine peaks (Fig. 61, the histidine peak (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 phenylalanine peak (Fig. 121, the two major threonine peaks (Fig. 15), and the major valine peak (Fig. 18). The only example of a displacement in which the vegetative peak elutes early (marked with a V on the figure) is the second lysine peak (Fig. 10). The displacement in the second tyrosine peak (Fig. 17) was not reproduced in all runs and is not marked on the figure. Reproducibility of RPC-5 Chromatographic Profiles -The performance of the column was periodically monitored by passing Escherichia coli K-12 tRNA (SchwarziMann), aminoacylated with [Hlleucine, through the column, and comparing the elution profile with published results (28). A typical profile for E. coli leucyl-tRNA is shown in Fig. 19. Columns were repacked when resolution began to deteriorate (usually after about 25 column runs). RPC-5 chromatographic profiles of Dictyostelium tRNA are summarized in Table III. Only those profiles which showed sufficient resolution are included in the table. As indicated in the table, profiles were generally highly reproducible. Although individual tRNA preparations gave consistent profiles, however, some differences between tRNA preparations from the same stage were occasionally observed. For example, the quantitative differences in the two major glycine peaks (Fig.

done by multiplying the ratio of the raw data (first column value/second column value) by the ratio for total acceptance (78111026). These adjusted ratios show that within an error of about 15% there are no differences in amino acid acceptance by these preparations. RPC-5 Profiles of Aminoacyl-tRNAs from Growth and Development - Developmental tRNA, aminoacylated with :Haminoacid using developmental synthetase, and vegetative tRNA, aminoacylated with C-aminoacid using vegetative synthetase, were initially co-chromatographed with a steep gradient of 0.25 to 1.0 M RPC-5 buffer (100 ml). This initial run served two purposes: (a) it indicated the salt concentration elution range of the aminoacyl-tRNAs and (6) it increased the peak heights of small peaks, making them easier to detect. Independently isolated tRNA preparations were then aminoacylated, but this time the IX-aminoacid and vegetative synthetase were used with developmental tRNA and the Haminoacid and developmental synthetase were used with the vegetative tRNA. These aminoacyl-tRNA preparations were combined and run with a very shallow gradient using the previously determined salt concentration elution range. If there were any inconsistencies between these two runs or if any aspects of the profiles remained to be clarified, further combinations of label and synthetase were used with different tRNA preparations. In all, up to five vegetative and three developmental tRNA preparations were used for a particular aminoacyl-tRNA. Recovery of labeled material from the columns always exceeded 90%. Figs. 2 to 18 are representative profiles of aminoacyl-tRNAs from growth and development. Reproducible quantitative differences, marked with a K on the figures, are found for some amino acids. The most dramatic quantitative differences are found in the profiles for asparaginyl-tRNA (Fig. 31, aspartyltRNA (Fig. 4), and tyrosyl-tRNA (Fig. 17). The only other reproducible quantitative differences are in minor peaks: a minor peak for lysine (Fig. lo), the final small peak for methionine (Fig. ll), the fourth proline peak (Fig. 131, three minor peaks for serine (Fig. 14), the minor peak for threonine (Fig.

ARGININE 4

2io FRACTION FRACTION

360

3bo FRACTION

FIG. 2 (lefi). RPC-5 chromatographic profile of arginyl-tRNA from vegetative (ueg) and developing (deu) cells. Recovered from the column were 108,000 cpm of developmental [H]tRNA and 18,000 cpm of vegetative [CltRNA. FIG. 3 (center). RPC-5 chromatographic profile of asparaginyltRNA from vegetative (ueg) and developing (deu) cells. Recovered

from the column were 204,000 cpm of developmental LHltRNA and 15,000 cpm of vegetative [Y!ltRNA. FIG. 4 (right). RPC-5 chromatographic profile of aspartyl-tRNA from vegetative (ueg) and developing (deu) cells. Recovered from the column were 169,000 cpm of vegetative [HltRNA and 64,000 cpm of developmental [WltRNA.

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tRNA.s

of

Dictyostelium

discoideum

699

12 0

GLUTAMIC

ACID

L\. 160 FRACTION FRACTION 160 200 FRACTION 220

5 (left). RPC-5 chromatographic from vegetative (ueg) and developing (dew) column were 226,000 cpm ofdevelopmental of vegetative [ C]tRNA. FIG. 6 (center). RPC-5 chromatographic from vegetative (ueg) and developing (deu)
FIG.

profile of glutamyl-tRNA cells. Recovered from the IHJtRNA and 59,000 cpm profile of glycyl-tRNA cells. Recovered from the

column were 221,000 cpm of vegetative [CltRNA. FIG. 7 (right). RPC-5 from vegetative (ueg) and column were 302,000 cpm of vegetative [CltRNA.

of developmental

l:HltRNA

and 42,000 cpm

chromatographic profile of histidyl-tRNA developing (deu) cells. Recovered from the of developmental [:HJtRNA and 22,000 cpm

3 7 % 1 n
IV.0

ISOLEUCINE

LEUCINE

Ij!

LYSINE

j.
$06

j5 FRACTION

scl FRAi;ON

Ii0

FIG. 8 (left). RPC-5 chromatographic from vegetative (ueg) and developing (deu) column were 427,000 cpm ofdevelopmental of vegetative I CltRNA. FIG. 9 (center). RPC-5 chromatographic from vegetative (ueg) and developing (deu)

profile of isoleucyl-tRNA cells. Recovered from the IHltRNA and 76,000 cpm profile of leucyl-tRNA cells. Recovered from the

column were 80,000 cpm of developmental lHltRNA and 56,000 cpm of vegetative LITltRNA. FIG. 10 (rLgght). RPC-5 chromatographic profile of lysyl-tRNA from vegetative (ueg) and developing (deal cells. Recovered from the column were 94,000 cpm of vegetative [HltRNA and 33,000 cpm of developmental L1CltRNA.

6) and the second methionine peak (Fig. 11) were not found in other runs. Also, although quantitative differences in the first methionine peak (Fig. 11) were reproduced in all runs, when two vegetative methionyl-tRNA preparations were co-chromatographed, they showed quantitative differences of similar magnitude. Because of results such as these, quantitative differences of less than 20%, especially in minor peaks, were extremely difficult to substantiate. Although such differences might exist in viuo, they were beyond the resolution of this analysis. In addition, some minor peaks were encountered in which the peak tube represented less than 0.1% of the total

recovered counts. Peaks in this size range could not be accurately analyzed. It is impossible for us to equate the number of peaks found for a given aminoacyl-tRNA with its number of isoaccepting species. Besides the possibility that some peaks are different modifications of the same gene product, some may also be due to the presence of aggregates, tRNA fragments, or amino acid acceptors other than tRNAs (15). Since the purpose of this analysis was only to screen for differences between vegetative and developmental tRNA, these other possibilities have not as yet been pursued.

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700

tRNAs

of Dictyostelium

discoideum

3
METHIONINE 4

FRACTION

FIG. 11 (left). RPC-5 chromatographic profile of methionyl-tRNA from vegetative (ueg) and developing (den) cells. Recovered from the column were 113,000 cpm of developmental L:!H]tRNA and 46,000 cpm of vegetative [YS]tRNA. FIG. 12 (center). RPC-5 chromatographic profile of phenylalanyltRNA from vegetative (ueg) and developing (deu) cells. Recovered

from the column were 357,000 cpm of developmental [HltRNA and 66,000 cpm of vegetative [TltRNA. FIG. 13 (right). RPC-5 chromatographic profile of prolyl-tRNA from vegetative (ueg) and developing (deu) cells. Recovered from the column were 125,000 cpm of developmental L3HltRNA and 4000 cpm of vegetative [T]tRNA.

I2

SERINE

SO

THREONINE
3

c4

F2 ,

TRYPTOPHAN

-jOS

1
; 0.6 0 d

100

150

200

250

300

7b

90

II'0

FRACTION
FIG.

FRACTION

FRACTION

14 (left). RPC-5 chromatographic profile of seryl-tRNA from vegetative (ueg) and developing (deu) cells. Recovered from the column were 184,000 cpm of vegetative [H]tRNA and 24,000 cpm of developmental [CltRNA. FIG. 15 (center). RPC-5 chromatographic profile of threonyltRNA from vegetative (ueg) and developing (deu) cells. Recovered
DISCUSSION

from the column were 212,000 cpm of vegetative 24,000 cpm of developmental [T]tRNA. FIG. 16 (right). RPC-5 chromatographic profile tRNA from vegetative (ueg) and developing (deu) from the column were 390,000 cpm of vegetative 19,000 cpm of developmental [TltRNA.

LRHltRNA

and

of tryptophanylcells. Recovered 13HltRNA and

Comparison of Vegetative and Developmental tRNA for Acceptance of Amino Acids -We have found no major difference between vegetative and developmental tRNAs in their level of aminoacylation with each of the 17 amino acids investigated (Table II). In contrast, significant changes in tRNA populations do occur in several other developing systems, including the silk gland, lens, and reticulocyte (4, 5). All of these systems, however, are characterized by differentiation to produce large quantities of a few proteins having an unusual amino acid content, and in each case the change in tRNA

populations parallels the change in amino acid composition of the proteins being made. Hence the concentrations of the tRNAs are believed to adjust to permit efficient synthesis of the specialized proteins (4-6, 31). Since D. discoideum synthesizes many different proteins throughout development, it is not surprising that no major readjustment in levels of amino acid acceptance occur. However, these results do not preclude there being important developmental changes in the tRNAs; there could be qualitative changes in the tRNA molecules, or there could be compensating quantitative changes within the subpopulation of isoaccepting species for a given amino acid.

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tRNAs
TYROSINE

of Dictyostelium

discoideum
VALI NE

701

I K
.L. 245 270 295 320 345
; K I

50

FRACTION FIG. 17 (left). RPC-5 chromatographic profile of tyrosyl-tRNA from vegetative (ueg) and developing (deu) cells. Recovered from the column were 218,000 cpm of developmental 1:HltRNA and 26,000 cpm of vegetative 1CltRNA.

I,_____ 1 a0 110 FRACTION

3 1
140 170

FIG. 18 (right). RPC-5 chromatographic profile of valyl-tRNA from vegetative (ueg) and developing (deu) cells. Recovered from the column were 317,000 cpm of developmental 1:HltRNA and 19,000 cpm of vegetative 1CltRNA.

T I 0.0 L r 0.7 I2 w

062 5 I0.5: 0-J

FRACTION 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 (200 ml) was used. Recovered from the column were 186,000 cpm.
FIG.

The results of analysis of the tRNAs on reversed phase columns, discussed below, indicate changes do occur. Quantitative Differences between Aminoacyl-tRNAs from Growth and 18 H of Development-Quantitative differences between aminoacyl-tRNAs eluted from RPC-5 columns are found in both major and minor peaks. The most dramatic quantitative differences are found in the profiles for only three aminoacyl-tRNAs: asparaginyl-tRNA (Fig. 31, aspartyl-tRNA (Fig. 4), and tyrosyl-tRNA (Fig. 17). Curiously, these tRNAs, along with histidyl-tRNAs, are the only ones for which major

quantitative differences were observed during Drosophilia development. (32). Since it was shown in that system that differences in a Q base modification were responsible for the shifting of the aminoacyl-tRNAs from one peak to another, it is possible that a similar post-transcriptional change, rather than quantitative differences in different gene products, may be responsible for the analogous differences which we observe. Some of the differences in minor peaks may be due to the presence of mitochondrial tRNAs. In general, however, differences in minor peaks accounted for a very small number of the actual changes observed. The quantitative difference in the final small peak of methionine (Fig. 11) is interesting in light of the restriction in protein synthesis which occurs early in Dictyostelium development (33, 34). We do not as yet know when this quantitative change takes place or if the peak represents a minor cytoplasmic initiator tRNA. Displacement of Peaks of Aminoacyl-tRNAs -The most common class of differences between aminoacyl-tRNAs from growth and development is the displacement of peaks from one stage with respect to the other. This class of differences is highly reproducible for all independent tRNA preparations studied and does not depend on either the label utilized or the source of the synthetase in the aminoacylation reaction. This type of change is apparent for peaks corresponding to 11 out of the 17 amino acids studied. In many cases, the displacement is in the major peak, and in all but one case, the developmental tRNA elutes ahead of the vegetative tRNA. In most instances, a single peak for each amino acid is displaced. Independent confirmation of peak displacements for three amino acid peaks has been obtained on RPC-3 columns.: Preferential degradation is not a likely cause for the peak displacements observed. If there were degradation, it would have to reside in the tRNA preparations themselves since displacements did not change when stage-specific synthetases were interchanged in the aminoacylation reactions for the RPC-5 column runs. Such preferential degradation of the 3 M. Brenner, unpublished observations.

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702

tRNAs
Summary

of Dictyostelium
TABLE

discoideum profiles
in Given
4

III

of RPC-5

chromatographic % Total

Amino Acid

No. of rations V%
4

tRNA prepaanalyzed De"

3

No. of columns T"llS

Peak

zt S.D.a
5 6 7

1
V D 68.3 71.1 + 4.4 i 1.7 31.7 28.9 1.11 30.6 51.8 0.59 35.0 7.0 10.36 3.1 3.5 0.99

2 + 4.4 T 1.7 F 0.15 + 5.1 7 1.4 67.0 41.2 1.63

A%

8
3

0.96
2.4 7.0

z 0.06
+ 0.4

v
3 2 D

R ASP
3 2 3 V D

0.35
65.0 93.0

+ 1.1 T - 0.03
+ 3.0 7 3.8

7 - 0.11
+ 3.0 7 3.8 T - 6.55 + 0.4 + 1.4 T 0.30 + 0.5 ? 9.0

+ 5.4 7 0.4 z 0.15

R
Glu 2 2 2 D

0.70
9.1 12.2

5 0.03
+ 4.2 ? 1.2

R
V GUY

0.73
29.7

i- 0.28
+ 0.5

87.9 84.4 1.04

+ 4.6 i 2.5 T - 0.02

1
4

D R (one v D R v D R V D R

28.6 1.15 peak

3.0 2.8

+ 9.0 + - 0.35 only)

+ 0.6 T 0.9

70.3 71.4

1.00

T - 0.12

His Ile

1.35 14.9 15.9 1.04 10.0

8.4

3 0.39 + 1.3 7 1.2 z 0.03 + 5.6

+ 4.4

8.5 12.7 0.70 53.4 54.7 0.99

+ 0.5 T 1.5 T - 0.12 + 2.0 + 2.6 T - 0.03

78.0 75.5 1.03 8.9 6.0 1.04

+ 0.7 7 1.4

3.2 2.6

+ 0.4 + 0.1

5.3 4.6

+ 0.3 + 0.3

2.1 2.4

? 0.01 + 1.6 + 0.7 7 - 0.08 + 2.6 T 1.5 T - 0.12 + 4.0 i 1.5 + - 0.08

1.25
18.2

7 - 0.15
+ 3.1

1.18
6.5 5.8 1.14

T 0.12 + 0.8

0.91
5.7 4.7

+ 0.1 ? 0.1 z 0.09

+ 0.6 7 0.4

LL?U

12

18.2
1.03

+ 4.1 7 - 0.08
+ 4.5 T 2.6 IO.05 + 0.6

T 1.0 T - 0.13

1.10

+ - 0.15

27.1
28.3 0.96 33.8 28.1 1.22 16.7 12.8

+ 1.3
7 1.7 z 0.02 + 4.5 + 1.3 3 0.21 + 3.0

LYS

1.16
4.2 6.7

+ - 0.12
+ 0.2 + 1.0

9.6 14.1 0.66 58.8 64.9 0.87

55.7 52.7 1.05 3.3 0.3 8.17

v
Met 5 3 8

D R V D R v D R v D
R v

+ 0.1
+ - 1.42

0.76 83.3 87.2 0.96 11.3 11.3 1.19 1.3

0.4 5.55 2.7 5.0 0.54

T - 0.10 + 3.0 + 1.0 + - 0.02 + 5.4 + 5.4 T - 0.18 + 0.7

T 0.2 + - 4.55 + 0.6 i 1.3 z 0.03

Phe

1.28 71.8
74.9 0.96 87.3 93.5 0.93 32.8 36.1 0.91 72.3 60.8 1.13 33.1 18.4 1.80 95.3 96.3 0.99

+ 1.0 E 0.15 +13.5

Tl4.4 9.2 8.8 1.08 5.4 1.9 2.89 64.9 58.9 + 5.1 T 5.2 T - 0.07 + 0.7 7.9 5.9 1.50 + 3.1 i 3.2 T 0.29 -

x 0.01
+ 2.2 i 0.3 T - 0.02 + 3.3 7 2.6 + - 0.03 +11.9 T 8.6 + - 0.06 + 2.0 7 1.3 T - 0.17 + 1.8 + 2.9

1.4
0.7 2.07

+ 0.3
+ 0.3 T - 0.37

1.0
0.3 3.08

+ 0.2
T 0.0 T - 0.93

SeII

T 0.1
? - 0.54 + 3.6 7 3.8

2.7 2.5 1.12

+ 0.7 i 0.9

1.3 0.7

+ 0.3 T 0.0

T - 0.12

1.82

z 0.38

Thr

D
R

1.10
4.6 15.0 0.30

i- 0.01
+ 1.4 T 1.2 T 0.07 5.6 l.lT 7.16 + 0.4 0.4 z 2.34 3.7 0.7 5.20 + 1.4 7 0.3 i- 0.64

TOP

V D
R

23.1 24.2
0.97

+10.6 T 7.4
? - 0.18

TY~

V D R

41.6 77.1 0.57

2.3 0.8

+ 2.8 7 2.1 i- 0.03

+ 0.7 + 0.2

17.5 + 1.6 3.2 F 0.5 5.53% 1.22 3.4 4.3 0.75 + 1.6 + 1.7 z 0.07

v
Val
2 2 2 D

2.95

? - 0.28

IO.01

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

tRNA preparations is unlikely because (a) vegetative and developmental tRNAs were prepared in identical fashion, (b) displacements were consistent for five different vegetative and three different developmental tRNA preparations, (c) gel electrophoretic analyses of RNA preparations showed little evi-

dence of degradation, (d) rates and extents of aminoacylation were identical for tRNAs from both stages, even when stagespecific synthetases were switched, suggesting (but not proving) that synthetase recognition was identical for both, (e) only certain peaks were affected, and (f) heating of vegetative

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tRNAs

of Dictyostelium

discoideum

703

tRNA, to 80 for 10 min (35) did not cause any peaks to be displaced. We cannot, of course, completely rule out the possibility of preferential degradation without doing fingerprint analyses. Nature ofDifferences in Aminoacyl-tRNAs between Growth and Development-Although some of the peak displacements may result from quantitative differences within a heterogenous peak, others are probably due to differences in posttranscriptional modification between vegetative and developmental tRNA. Levels of acceptance of aminoacyl-tRNAs from both stages are extremely similar. In addition, in most of the cases in which a peak displacement is observed, the overall profile and the heights of the specific peaks in question are also strikingly similar. Even the differences observed in the aspartic acid, asparagine, and tyrosine profiles may represent differences in modification, as was demonstrated for changes in these same tRNAs during Drosophilia development (32). The fact that displaced peaks almost always have the developmental tRNA eluting ahead of the vegetative tRNA further suggests that a common change or a very small number of changes in modification of these tRNAs are occurring during Dictyostelium development. In the his T mutants of Salmonella, for example, it was shown that a lack of pseudouridine in the anticodon loop, caused by the absence of a single modifying enzyme activity, resulted in at least 10 peak displacements, corresponding to several different amino acids. All peak displacements were in the same direction (8, 9, 36). Peak Displacements Are Not Unique to Dictyostelium Development -The observation that there were peak displacements for most of the amino acids tested prompted us to reexamine published profiles of aminoacyl-tRNAs in other developing systems to see if peak displacements could also be detected. Such examination suggests that displacements are present. Some examples include the lysyland methionyltRNAs of developing brine shrimp (Ref. 37, p. 30), the seryltRNA of milkweed bugs undergoing embryogenesis (Ref. 38, p. 127), and the histidyl-tRNA from different tissues during mouse development (Ref. 39, pp. 68-69). Peak displacements are therefore probably not unique to Dictyostelium development and should be considered as examples of unresolved heterogeneity, possibly due to differences in post-transcriptional modification. Relationship of Differences to Translational Control -Because of the central position occupied by tRNAs in protein synthesis, any analysis of tRNA changes during development must consider the postulated role of tRNA in translational control (1). The possibility that some of the differences we observe may represent differences in Q base modification in the anticodon loop is of interest in this regard. In preliminary work with an in vitro protein synthesizing system from wheat germ programmed with Dictyostelium vegetative mRNA, however, tRNAs from both stages appeared to function equally well in their ability to stimulate incorporation of radioactive amino acids into hot trichloroacetic acid-precipitable material. This approach is presently being extended by comparing on gels products synthesized in vitro with stagespecific tRNAs. The possibility that some of the tRNA changes affect functions other than translation should also be considered. The analogy of the peak displacements observed here to those resulting from the his T mutation in Salmonella, which is known to affect gene transcription (8, 9, 141, has already been pointed out.

Acknowledgments -We are extremely grateful to Paula A. Palatnik for help with the computer analysis of the data and to Larry E. Fields and Macy Koehler for help with the experimental work. REFERENCES 1 Sueoka. N.. and Kane-Sueoka. T. (1970)Proe. Nucleic Acid Res. Mol. siot. 10, 23-55 2 Littauer. U. Z.. and Inouve. Reu. Biochem. 42, H. (1973) Annu. 439-470 3 Scherberg, N. H., and Weiss, S. R. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1114-1118 4. Garel, J-P. (1974) J. Theor. Biol. 43, 211-225 5. Smith, D. W. E. (1975) Science 190, 529-535 6 Garel, J-P. (1976) Nature 260, 805-806 7. Ilan. J.. and Ilan. J. (19751 Curr. TOD. Deu. Biol. 9. 88-136 8. Cortese; R., Landsberg, R., Vender Haar, R. A., Umbarger, H. E., and Ames, B. N. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1857-1861 9. Rizzino, A. A., Bresalier, R. S., and Freundlich, M. (1974) J. Bacterial.117. 449-455 10. Lodish, H. F., Jacobson, A., Firtel, R., Alton, T., and Tuchman, J. (19741 Proc. Natl. Acad. Sci. U. S. A. 71, 5103-5108 11. Jacobson, A., Firtel, R. A., and Lodish, H. F. (197415. Mol. Biot. 82, 213-230 12. Brenner, M., Tisdale, D., and Loomis, W. F., Jr. (1975)Exp. Cell
Res. 90, 249-252

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W. F.. Jr. (1975) Dictvostelium discoideum: A DevetooSystem, Academic Press, New York Brenner. M.. and Ames. B. N. (1971) in Metabolic Pathways (Vogel, H.J., edj Vol. 5, pp. 349-387, Academic Press, New York Soffer, R. L. (1974)Adu. Enzymol. Relat. Areas Mol. Biol. 40,91139 Marin. F. T. (1976) Dev. Biol. 48. 110-117 Loomis, W. F., Jr. (1971) Exp. Cell Res. 64, 484-486 Coccuci. S.. and Sussman M. (1970) J. Cell Biol. 45, 399-407 Watts, D. J., and Ashworth, J. M. (1970) Biochem. J. 119, 171174 Brenner, M., and Ames, B. N. (1972) J. Biol. Chem. 247, 10801088 Sarin, P. S., and Zamecnik, P. C. (1964) Biochim. Biophys. Acta 91, 653-655 Layne, E. (1957) Methods Enzymol. 3, 450-451 Kissane, J. M., and Robins, E. (1958) J. Biol. Chem. 233,184-188 White, B. N., Tener, G. M., Holden, J., and Suzuki, D. T. (1973) Deu. Biol. 33, 185-195 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Rubin, I. B., Mitchell, T. J., and Goldstein, G. (1971) Anal. Chen. 43, 717-721 Bonnett, J., and Ebel, J. (1972) Eur. J. Biochem. 31, 335-344 Kelmers, A. D., and Heatherly, D. E. (1971) Anal. Biochem. 44,
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29. Roe, B., Marcu, K., and Dudock, B. (1973) Biochim. Biophys. Acta 319, 25-36 30. Yund, M. A., Yund, E. W., and Kafatos, F. C. (1971)Biochen. Biophys. Res. Commun. 43, 717-722 31. Delaney, P., and Siddiqui, M. A. Q. (1975) Dew. Biol. 44,54-62 32. White, B. N., Tener, G. M., Holden, J., and Suzuki, D. T. (1972) J. Mol. Biol. 74, 635-651 33. Tuchman, J., Alton, T., and Lodish, H. F. (1974) Deu. Biol. 40, 116-128 34. Lodish, H., Alton, T., Margolskee, J., Dottin, R., and Weiner, A. (1975) in Developmental Biology: Pattern Formation, Gene (McMahon, 383. W. A. Beniamin. 35. Adams, A., and Zachau; 36. Singer, C. E., Smith, G.
Nature New 23. 23-25 Biol. 238, Regulation, lar Biology ICN-UCLA Symposium on Molecular and Cellu-

D., and Fox, C. F., eds) Vol. 2, pp. 366Menlo Park, Ca. H. G. (1968)Eur. J. Bzochem. $556-558 R., Cortese, R., and Ames, B. N. (1972)
72-74

37. Bagshaw,
Biol.

J. C., Finamore,

F. J., and Novelli,

G. D. (1970) Dew.

38. Smith, R,L., and Forest, H. S. (1973) Deu. Biol. 33, 123-129 39. DeLeon, V., Yang, W. G., and Sirlin, J. L. (1975) Differentiation 4, 65-72

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