Quantitative Measurement of Rates of 5S RNA and Transfer RNA Synthesis in Sea Urchin Embryos

~

Differentiation

Differentiation 15, 97-105 (1979)

0 Springer-Verlag I979

Quantitative Measurement of Rates of 5s RNA and Transfer RNA Synthesis in Sea Urchin Embryos ANNE F. O’MELIA’ Department of Biochemistry, Louisiana State University Medical Center, 1100 Florida Avenue, New Orleans, Louisiana 701 19 USA

Embryonic differentiation is believed to be due to a programmed expression of genes, which includes their time of activation, sequence of appearance, and amount transcribed into the immediate gene product, RNA. Differential synthesis of the major RNA classes, such as the ribosomal RNAs (28S, 18S, 5s) and transfer RNA (tRNA), characterizes many animal developing systems, including the sea urchin embryological system. Previous work has shown that the genes for 5s RNA and tRNA are active during early cleavage in sea urchin embryos. The present study focused on quantitatively measuring and comparing the rate of 5s RNA and tRNA synthesis in cleavage, early blastula, and early pluteus embryos of Arbacia punctulata. At each stage, embryos were labeled for 3 h with [83Hl-guanosine. Total cellular RNA was extracted using the cold ( 4 O C)-phenol-sodium dodecyl sulfate method and purified (LiCl-soluble) RNA preparations were fractionated by electrophoresis on 1Wo polyacrylamide gels. The amount of 5s RNA and tRNA synthesized at each stage was calculated from the radioactivity coincident with the 5s RNA and with the tRNA absorbance peaks (Az6,, ,,,) on each gel, from the known guanosine monophosphate (GMP) compositions of sea urchin 5s RNA and tRNA and from the average specific radioactivity of the GTP precursor pool during each 3 h labeling period. The results showed that on a per embryo basis the rates of 5s RNA and tRNA synthesis increased slightly (about 1.4-fold) from cleavage through pluteus stages, while on a per cell basis the rates declined severalfold (about %fold) during embryogenesis. The rates of 5s RNA and tRNA synthesis determined here parallel previously-reported levels of RNA polymerase 111 in sea urchin embryos, suggesting that cellular levels of RNA polymerase I11 may exert some positive control over 5s RNA and tRNA synthesis during sea urchin embryogenesis.

Introduction

Significant changes in RNA metabolism have been described during differentiation of the sea urchin embryo. Numerous studies have measured and compared the rates of synthesis of several major classes of high molecular weight RNA, such as the nucleolar ribosomal RNAs (rRNAs) [l-41, the heterogeneous nuclear RNAs (HnRNAs), and messenger RNAs (mRNAs) [5-111. In contrast, the synthesis of low molecular weight RNAs, such as 5s RNA and transfer RNA 1 Present address: Department of Biology, George Mason University, 4400 University Drive, Fairfax, Virginia 22030, USA

(tRNA), has not been investigated extensively during sea urchin embryogenesis. Early studies [12, 131, employing sucrose density gradient centrifugation and base composition analysis, failed to detect the synthesis of tRNA before the mesenchyme blastula stage. Any label seen in the 4s RNA region of sucrose gradients was believed to be due to the pCpCpA end-terminal turnover of pre-existing tRNA molecules. More recently, the use of appropriate RNA precursors and sensitive fractionation procedures, combined with experiments designed to discriminate between end-terminal labeling of tRNA and de novo synthesized tRNA, permitted the direct demonstration of newly-synthesized tRNA and 5s RNA in cleaving sea urchin em-

0301-468 1/7 9/00 15/009 7/ $ 0 1.80

98 bryos 114, 151. The purpose of the present study was to

quantitatively measure and compare the rates of synthesis of 5 s RNA and of tRNA during several stages of early sea urchin development. Methods Culturing and Labeling of Sea Urchin Embryos Arbacia punctulata embryos were grown at 3 x lo4 embryos per ml in MBL-formula artificial sea water [161, containing appropriate concentrations of antibiotics 114, 151 (ASW + P/S). At the desired developmental stage, embryos were collected and resuspended in ASW + P/S, containing [8-3H]-guanosine at a concentration of 0.46 pM [Schwarz/Mann, Orangeburg, New York; 6.5 Ci mmol-', 3 pCi ml-I). The addition of radioactive nucleoside had no observable effect on embryonic growth and development, as judged by comparison with embryos grown simultaneously in unlabeled medium. The number of embryos per culture and the number of cells per embryo at each stage were determined by the method of Hinegardner 1171. Guanosine was chosen as the RNA precursor because: (1) 5s RNA and tRNA are G + C-rich RNA species, (2) previous work [ 5 , 111 has shown that the )H-GTP precursor pools in sea urchin embryos can be labeled to moderate specific activities, and (3) guanosine is not easily converted to other nucleotides in sea urchin embryos [5,181 nor will it participate in the pCpCpA turnover of tRNA [14, 151. Therefore, guanosine may be used to directly measure RNA synthesis in this material. Extraction and Fractionation of 5s RNA and tRNA

Sea urchin embryos at each of four stages of development [cleavage (3 h), early blastula (8 h), gastrula (13 h), and early pluteus (21 h)l were labeled for 3 h with [8-3H1-guanosine.Total cellular RNA was extracted by the cold (4' C)-phenol-sodium dodecyl sulfate method and was purified further by extensive DNAase and pronase digestion [14, 151. Total soluble RNA (low molecular weight RNA, including 5s RNA and tRNA) was prepared by treating each total cellular RNA preparation with high salt (2.0 M LiCl) [191, as described in detail elsewhere [ 141. Reportedly this procedure selectively and quantitatively precipitates high molecular weight RNA 191 and significant amounts of precursor-tRNA [20]. The latter has been shown to interfere with the resolution of newly-synthesized 5s RNA and tRNA (4s) on polyacrylamide gels [15, 20, 211. Purified soluble RNA preparations were fractionated by electrophoresis on 10% polyacrylamide gels [22]. as described previously [14, 151. Gels were scanned at 26011x11 in a Gilford recording spectrophotometer. Each gel was sliced into 1.0 mm sections and each section was solubilized in 0.3 ml fresh superoxol and 1.5 ml NCS (Amersham/Searle, Arlington Heights, 111.). After the addition of 10 ml toluene-POPOP-PPO scintillation fluid, radioactivity was measured at ambient temperature in a Beckman LS-3145T liquid scintillation counter using the external standard and experimentallyestablished quench curves. The recovery of tritium was 90% or greater. 5 s RNA and tRNA (4s) were identified by: (1) their electrophoretic mobility, which is inversely proportional to the logarithm of their respective molecular weight and (2) their coincident migration with internal standards (5s RNA and tRNA from Escherichia coli,

A. F. O'Melia: 5s RNA and tRNA Synthesis in Sea Urchins Miles Laboratories, Elkhart, Ind. and 14C-labeled5s RNA isolated from pluteus ribosomes). The in vivo methylation, using L-[14C-or 3H-methyll-methionine as precursor, of tRNA in embryos of A. punctulata [ 14, 151, under conditions where the [labeled-methyllgroups are not utilized for C,-units in purine biosynthesis, has been previously reported. In addition, the RNA species identified here as 5s RNA does not undergo methylation in vivo 114, 151. The lack of methylation of 5s RNA distinguishes it from most (if not all) other small molecular weight nuclear RNAs in sea urchin embryos 1231 as well as most potential RNA breakdown products produced during isolation procedures. Base Composition Analysis

An aliquot of each soluble RNA preparation was acid hydrolyzed into its 2'-, 3'-ribonucleotides by incubation in 1 N HCIO, at 25' C for 18 h. The hydrolysate was neutralized (pH 7-8.5) by the addition of 3 N KOH. After the KCIO, precipitate was pelleted by centrifugation, the supernatant was made 0.05 N in HCl and an aliquot was applied to a Dowex-50-H (Sigma Chemical Co., DeKalb, Mo.) column according to the method of Katz and Comb [241. One milliliter fractions were collected and read in a Beckman spectrophotometer at the wavelength of maximum absorption for each nucleotide: UMP (260 nm); GMP (257 nm); AMP (257 nm); CMP (279 nm). An aliquot (0.1 ml) of each fraction was dried on a Whatman Gf/A filter pad and was counted in toluene-based scintillation fluid at ambient temperature in a Beckman LS-3 145T liquid scintillation counter. Determination of the Specific Radioactivity of 3H-GTP

Embryos at cleavage (3 h), early blastula (8 h), and early pluteus (21 h) stages were labeled with 3H-guanosine. Measured aliquots were removed from each culture at 0-, 30-, 60-, 120-, and 180-min after isotope addition. Each aliquot was washed by diluting it twofold with ice-cold ASW + P/S, containing 0.1 mM unlabeled guanosine, before centrifugation. The washing procedure was repeated twice to ensure removal of exogenous label. Embryo pellets were frozen in dry-ice acetone and stored at -209C. The total acid soluble pool was extracted by homogenizing each embryo pellet twice in a Dounce homogenizer with 2-5 ml of 0.5 N HCIO, at 0' C for 10-20 min. Acid insoluble material was removed by centrifugation at Oo C for 15 min at 10,000 g. The acid soluble supernatant was adjusted to pH 7.0-8.5 by the stepwise addition of 3 N KOH and 1 N KOH. After 30 min at 0' C, the KC10, precipitate was removed by centrifugation at 00 C for 15 min at 10,OOO g. Guanosine triphosphate (GTP) was isolated from each acid-soluble extract by methods similar to those of Kijima and Wilt [51 and Galau et al. [ill with some modifications in elution procedures. Small columns (0.5 x 5.0 cm) of Dowex-1-formate [AG-1-X8, 200-400 mesh (formate form); BioRad Laboratories, Richmond, Ca.] were formed in disposable Pasteur pipets plugged with glass wool. After washing each column with water, a sample (in 0.05 N NH,OH) was applied and the column was washed with 10ml of distilled water. Nucleotides were eluted by gravity flow with 10 ml of solution A (IN formic acid, 0.5 N ammonium formate), 12 ml of solution B (IN formic acid, 0.75 N ammonium formate), 16 ml of distilled water, and 15 ml of 0.5 N HCI. A column which was not loaded with acid soluble material was run to provide spectrophotometric blanks. Figure la shows the fractionation of several standard nucleotides on Dowex-1-formate columns. Only GTP is found in fractions

99

A. F. OMelia: 5s RNA and tRNA Synthesis in Sea Urchins

Fig. 1. Fractionation of nucleotides on Dowex-lformate columns. (a) Elution pattern of some standard nucleotides from Dowex-1-formate columns as described in methods section. GMP, GDP, GTP, ATP, UTP (Sigma Chemical Co., DeKalb, Mo.) were run individually on columns with identical elution schedules. In addition, GMP, GDP, GTP were run in various combinations (GMP, GDP, GTP; GMP, GDP; GMP, GTP; GDP, GTP; GMP, GTP) on the same column. A and B represent the time of addition of solutionA and solutionB as described in methods. (b) The elution pattern of the total acid-soluble fraction from early blastulae labeled for 120 min with [8-3H]-guanosineat a concentration of 0.46 pM

120

80 a

g %x 4

40

f 0

10

20

u)

M

Amount of Effluent (ml. 1

eluted with 0.5 N HCl, and the recovery of GTP is always 90% or greater. When acid-soluble extracts from labeled embryos, such as that of blastulae labeled with 0.46 pM [8-3H]-guanosinefor 120 min (Fig. lb), were fractionated on Dowex- 1-formate columns, tritiumlabeled material with absorbance at 260 nm eluted from the column as if it were GTP. Identification of this material as GTP was made by pooling the fractions which had been eluted with 0.5 N HCl, lyophilizing them to dryness, dissolving the lyophilizate in 50 p1 of distilled water, and spotting the sample (in parallel with known concentrations of standard nucleotides) on Polygram CEL 300 PEI/UV 254 plates impregnated with fluorescence indicator (Brinkmann Instruments, Inc., Westbury, NY). Chromatograms were prewashed and developed essentially as described elsewhere [25, 261. The positions of standard nucleotides and of sample GTP were determined using a short wave ultraviolet lamp. Spots corresponding to sample GTP and to standard GTP were cut out, eluted for 30min with 0.5 ml of 0.7 M MgCl,, 20 mM Tris (pH 7.4), and centrifuged for 10 min at 4,000 g.The concentration of GTP was determined by its absorbance at 260 nm using a molar extinction coefficient of 11.6 x lo3. Isolated sample GTP had similar spectral ratios to those of standard GTP. Sample GTP radioactivity was determined by dissolving a 50 p1 aliquot in 10 ml Triton-X-100-Toluenemixture (3 : 7) containing 5 g PPO and 0.3 g POPOP per liter and was counted at ambient temperature in a Beckman LS-3145T liquid scintillation counter.

Rates of Accumulation of Newly-Made SS RNA and tRNA Previous work [141 has shown that when total cellular RNA, extracted from 3H-guanosine-labeled cleavage

embryos, was fractionated by electrophoresis on a 10% polyacrylamide gel, newly-synthesized 5s RNA was incompletely resolved from newly-made tRNA presumably due to the presence of labeled precursor-tRNA of intermediate size between 5s RNA and tRNA (4s) [20, 211. In contrast, when purified soluble RNA, isolated from the same total RNA preparation, was fractionated on a similar gel, de novo-synthesized 5s RNA was completely resolved from newly-made tRNA. Therefore, for comparative purposes, purified soluble RNA at each developmental stage was used for analysis. The possibility was considered that the embryos during the 3 h labeling period could convert some of the 3Hguanosine nucleotides into adenosine nucleotides, which could participate in the pCpCpA turnover of pre-existing tRNA molecules. To exclude this possibility, each purified soluble RNA preparation was acid hydrolyzed into its 2', 3'-ribonucleotides and the nucleoside monophosphates were separated on Dowex-50-H columns [241. Figure 2 shows the fractionation of ribonucleotides from cleavage embryos. The results show that all tritium label is present in GMP and no radioactivity is present in the AMP region of the column. Similar results were obtained using preparations from later developmental stages. Therefore, all 3H-radioactivityin exogenous guanosine which is incorporated into RNA passes through the 3H-GTP pool. In addition, base composition analysis of purified soluble RNA preparations at each stage showed that they were G + C-rich (58%-60%).

100

A. F. O’Melia: 5 s RNA and tRNA Synthesis in Sea Urchins _.

each developmental stage were fractionated by electrophoresis on 10% polyacrylamide gels, similar absorbance profiles (A26o,) were obtained. Therefore, the amounts of 3H-guanosine incorporated into newly-made 5s RNA and tRNA at each stage can be measured from the radioactivity profiles. Figure 3 is a comparison of polyacrylamide gel profiles of newly-synthesized 5s RNA and tRNA from cleavage, early blastula, gastrula, and early pluteus embryos of Arbacia punctulata. The results show that 5s RNA and tRNA are synthesized at all stages examined. Comparison of the rates of accumulation of newly-made 5 s RNA and tRNA on a per embryo basis (dpm/104 embryos/3 h; Tables 1 and 2, column 3) indicates that the accumulation of label in these newly-made RNAs increased more than 10-fold from cleavage to early blastula and increased more than 1.6-fold between early blastula and early pluteus stages of development.

1.2

1.0

.8 (279nml

0

2

4

6

8

10 12 Fractions

14

16

18

20

Fig. 2. Fractionation of 2’, 3’-ribonucleotides from ’H-labeled cleavage embryos on a Dowex-50-H column. Purified soluble RNA from cleavage embryos, labeled for 3 h with 0.46 pM 18-3HHl-guanosine, (same sample as that used in Fig. 3) was acid hydrolyzed and the component 2’, 3’-ribonucleotides were separated on a Dowex-50-H column [241

The RNA content of the sea urchin embryo remains fairly constant during early development [15, 27, 281. The total soluble RNA content, as determined by absorbance at 260 nm, similarly remained fairly constant (+ 5%). When similar amounts of soluble RNA (50 pg) at

The Behavior of the GTP Precursor Pool and the Specific Radioactivity of 3H-GTP

In order to compare the amount (grams) of 5s RNA and of tRNA synthesized at different stages of sea urchin embryogenesis, the average specific radioactivity of the 3H-GTP precursor pool was determined during each 3 h labeling period. Figure 4 shows the specific activities of 3H-GTP, measured at 0-, 30-, 60-, 120-, and 180-min after addition of exogenous isotope to cleavage, early blastula,

Table 1. Quantitative accumulation of newly made 5s RNAa

Labeling period (stage, hours after fertilization) Cleavage, 3-6 h

Average specific activity of GTP precursor podb (dprn/10-12 mol GTP) 25

5s RNA (dpm/104 embryosy

Accumulation of newly made 5s RNA g/embryo/hd

g/cell/he

mol/ceU/hf

52 (k 4)

6.95 x

3.48 x

5,237

.___

Blastula, 8-11 h

198

572 (k 44)

9.66 x 10-14

1.93 x

2,905

Pluteus, 21-24 h

391

1,150 (+ 113)

9.83 x lO-I4

1.23 x 10-l6

1,851

Calculations based on data of Figs. 3 and 4 Determined from the data shown in Fig. 4 by integrating the area over time in each curve and dividing by the total time elapsed (180 min) 5s RNA accumulation was determined from embryo counts per culture and from data of incorporation of [8’HI-guanosine as ’H-GMP into newly-synthesized 5s RNA (Fig. 3). The numbers in parentheses indicate the standard error of the mean for three or more determinations Calculated assuming that the GMP composition of 5s RNA is 32.9 mol % [30] Calculated using the cell number at each stage, determined by method of Hinegardner [171 Calculated assuming the molecular weight of 5s RNA is 4.0 x 104 a

A. F. O'Melia:

5s RNA and tRNA Synthesis in Sea Urchins

101

BLASTIJLA

PLUTEUS

6

5

r DISTANCE

5

6

7

M I G R A T E D CCMI

Fig. 3. Comparison of polyacrylamide gel profiles of newly-synthesized 5 s RNA and tRNA (4s) from cleavage (3 h), early blastula (8 h), gastrula (13 h), and early pluteus (21 h) embryos ofArbnciupunctuZutu. At each stage, embryos were labeled for 3 h with [8-3H]-guanosine(6.5 Ci mmol-I; 3 pCi d-l).Fqual amounts (50 pg) of purified soluble RNA et each stage were fractionated by electrophoresis on 10% polyacrylamide gels at 5" C (5 mA tube-') for 3 h. Positions of 5s RNA and of tRNA (4s) were determined by absorbance scanning at 260 nm and by use of internal standards. Because similar absorbance scans (A,,, ",.,) were obtained at each stage, only radioactivity data which has been corrected for background counts (dpm per gel slice) are plotted. In addition, only that portion of each gel [41 mm to 79 mml containing 5s RNA and tRNA (4s) is shown. Note changes in the scale of radioactivity as development proceeds

Table 2. Quantitative accumulation of newly made transfer (4s) RNA* Labeling period (stage, hours after fertilization)

Average specific activity of GTP precursor poolb ( d p d lo-'* mol GTP)

4 s RNA (dpm/104 embryos)c

492 (& 36)

Accumulation of newly made 4.9 RNA g/embryo/hd

7.62 x 10V3

g/ceWh'

mol/cell/hf

Cleavage, 3-6 h

25

3.81 x

91,745

Blastula, 8-1 1 h

198

6,320 (+ 181)

12.36 x

2.47 x

59,478

Pluteus, 21-24 h

391

10,500 (+ 395) 10.40 x

1.30 x

31,304

Calculations based on data of Figs. 3 and 4 Determined from data in Fig. 4 by integrating the area over time in each curve and dividing by the total time elapsed (180 min) Determined from data shown in Fig. 3, per Table 1 (c). Numbers in parentheses indicate standard error of the mean for three or more determinations Calculated assuming that the GMP composition of 4s RNA is 28.4 mol % [311 Calculated, per Table 1 Calculated assuming that the molecular weight of transfer RNA is 2.5 x lo4

a

e)

102

A. F. O'Melia: 5s RNA and tRNA Synthesis in Sea Urchins Blastula

500

0

30

60

120

180

Rg. 4. Specific radioactivity of 3H-GTP precursor pools. The specific radioactivity of the W G T P precursor pool was determined after 0-,30-, 60-, 120and 180-min incubation with 0.46 pM [%'HIguanosine (6.S Ci mmol-'; 3 pCi dP)at cleavage, early blastula, and early pluteus stages of Arbacia punetuluta development, as described in Methods. Vertical lines at each time point indicate the standard error of the mean; the numbers in parentheses represent the number of independent determinations at each time point

Time bin)

and early pluteus embryos. The 3H-dpm/pmol GTP was observed to rise rapidly to a maximum value at 60 min in cleavage embryos and at 30 min in early blastula and in early pluteus embryos. Because time points between 0-to 30-min and between 30- to 60-min were not explored in this study, the possibility that 3H-GTP reaches its maximum value between 30- and 60-min of incubation in cleavage embryos and between 0-to 30-min in early blastulae and plutei cannot be excluded. A rapid decline in 3H-GTP specific activity was found between 60- and 120-min in cleavage embryos and between 30and 60-min in early blastulae and early plutei. This decline is best explained by dilution of 3H-GTP with unlabeled GTP derived from the turnover of HnRNA and mRNA molecules [6, 10, 11, 291 synthesized before exogenous 3H-guanosine penetrated the embryo and became converted into 3H-GTP. At each stage, a steadystate level of 3H-GTP specific activity (pool equilibrium) was approached by the second to third hour of incubation with isotope. The average specific radioactivity of the 3H-GTP precursor pool at each stage was determined by integrating the area over time in each curve and dividing by the total time elapsed (180 min) [ 1, 21 (Tables 1 and 2, column 2). The results show that the average specific radioactivity of 3H-GTP was 25,198 and 391 d p d p m o l GTP, respectively, at cleavage, early blastula, and early pluteus stages. Therefore, the average 3H-dpm/pmol GTP increased about 8-fold between cleavage and early

blastula and increased about 2-fold between early blastula and early pluteus stages of development. Quantitative Measurement of Rates of 5.5 RNA and tRNA Synthesis

The quantitative accumulation of newly-synthesized 5s RNA and tRNA was calculated and compared at cleavage, early blastula, and early pluteus stages (Tables 1 and 2). The gram quantities of 5s RNA and tRNA synthesized per embryo per hour and per cell per hour were calculated from the radioactivity coincident with the absorbance peaks at 260 nm of 5 s RNA and tRNA on the 10% polyacrylamide gels (Fig. 3, column 3 in Tables 1 and 2), from the known GMP composition of sea urchin 5s RNA and tRNA classes 130, 311 and from the average specific radioactivity of 3H-GTP precursor pools (Fig. 4, column 2 in Tables 1 and 2). The calculations on a per cell basis assume that all nuclei are equally active in RNA synthesis. Comparison of the rate of 5s RNA synthesis at each developmental stage (Table 1) indicates that on a per embryo basis (column 4), the rate increased slightly (about 1.4-fold) between cleavage and pluteus stages. However, on a per cell basis (Table 1, columns 5 and 6), the rate of 5s RNA synthesis declined severalfold (about 3-fold) between early (cleavage) and later (pluteus) stages of sea urchin embryogenesis. Similar to the

103

A. F. O'Melia: 5 s RNA and tRNA Synthesis in Sea Urchins

rates of 5 s RNA synthesis (Table l), the overall rates of synthesis of tRNA (Table2) were found to increase slightly (about 1.4-fold) on a per embryo basis (column 4) and to decline about 3-fold on a per cell basis (columns 5 and 6). The rates of tRNA synthesis reported here are minimal rates as the contribution of newly-made precursortRNA was not measured and, therefore, was not included in the calculation. In addition, it should be noted that tRNA is a heterogeneous population of molecules, consisting of potentially more than 60 species. Therefore, the rates of total tRNA synthesis measured here would not reflect developmental changes in individual newly-made isoaccepting tRNAs [32-361. Discussion

The Behavior of the GTP Precursor Pool and the SpeciJic Radioactivity of 'H-GTP The present study is the first to measure the specific activity of 'H-GTP pools in Arbacia punctulata embryos. Furthermore, this is the first report to determine the specific activity of 'H-GTP during long labeling periods (3 h) at several stages of sea urchin embryonic differentiation. A previous report [ 111 extensively investigated the behavior of the 'H-GTP pools during longterm labeling of mesenchyme blastula-early gastrula embryos of Strongylocentrotus purpuratus. Both studies agree that, after the introduction of 'H-guanosine to the medium, the specific activity of the 3H-GTP pool rises rapidly to a maximum value and then decays over the next few hours in a characteristic manner [ll]. The major difference between the two reports concerns the time of maximum 'H-GTP specific activity, which occurs at 50 min in mesenchyme blastulae of S . purpuratus and at 30 min in early blastulae and early plutei of A . punctulata. This difference is best explained by the rapid timetable of development of A . punctulata (at 23" C ) compared to that of S . purpuratus (at 15" C).Different sea urchin species, which develop at different optimum temperatures, could be expected to have different rates of precursor transport and/or different rates of conversion of 'H-guanosine into 'H-GTP. In this report, the behavior of the 'H-GTP pools differs in cleavage embryos from that at later embryonic stages in that cleavage embryos exhibit an apparent 30min (or more) delay in reaching maximum specific activity. This delay could be due to differences in rate of transport of nucleoside into embryonic cells and/or differences in rate of metabolism of precursor to 'H-GTP.

In contrast, the differences in average specific radioactivities of 'H-GTP (Table 1 and 2, column 2) during embryonic differentiation are most likely due to differences in size of endogenous unlabeled GTP pools [371. Quantitative Measurement of Rates of 5s RNA and tRNA Synthesis The present study quantitatively measured rates of accumulation of newly-made 5 s RNA and tRNA. However, rates of accumulation of newly-synthesized molecules reflect both rates of synthesis and rates of degradation. If we assume that the newly-synthesized 5s RNA and tRNA are completely stable, then the rates of accumulation of these molecules are equal to their absolute rates of synthesis. Several lines of evidence suggest that newly-made 5s RNA and tRNA in sea urchin embryos are stable. Kinetic studies (personal unpublished results) indicate a constant rate of accumulation of 5s RNA and tRNA when embryos at each stage were labeled for 1.5-3.0 h; that is, when GTP pool equilibrium is attained. Reportedly, the constant rate of accumulation of a radioactive RNA indicates stability of that RNA [61. In addition, the half-life of mRNA (an unstable RNA class compared to more stable rRNAs and tRNA) in sea urchin embryos has been calculated to be between 5- and 6-h [lo, 111 and newly-made polysomal rRNAs (26S, 18s) have a decay rate constant of essentially zero [ill. A potential problem in measurements of rates of RNA synthesis is pool compartmentalization. However, the general agreement of data obtained by measurement of guanosine pools [ 5 , 111, or uridine pools [381, and of adenosine pools 1,21 on rates of RNA synthesis during sea urchin embryonic differentiation speaks against the possibility that the ribonucleotide triphosphate pools are compartmentalized in these embryos. The present study is the first to quantitatively measure rates of synthesis of any low molecular weight RNA during sea urchin development. The synthesis of RNA can be regulated by several possible positive and/or negative control factors, only one of which is the cellular level of RNA polymerase. Recent evidence suggests that RNA polymerase111 synthesizes 5s RNA and tRNA in eukaryotic cells [39-411. During sea urchin embryogenesis, the level of RNA polymerase I11 increases only slightly on a per embryo basis and declines severalfold on a per cell basis [381. The rates of 5s RNA and tRNA synthesis per embryo and per cell determined in this study parallel the previously-reported levels of RNA polymerase I11 [381, suggesting that cel-

104

lular levels of RNA polymerase 111 may exert some positive control over 5 s RNA and tRNA synthesis during the embryonic differentiation of the sea urchin. The mechanisms involved in this possible control presently remain to be explored. Acknowledgements: I wish to acknowledge support of this work by a PHS Biomedical Research Grant (5SO1 RR 05376-13) and by funds from Louisiana State University School of Dentistry. A portion of this work was presented in abstract form [421.

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