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Effect of dissociation of embryos of Urechis caupo on synthesis of ribosomal RNA
PRELIMINARY
NOTES
Effect of dissociation of embryos of Urechis caupo on synthesis of ribosomal RNA ENGSTROM,’ Department of Zoology, University of California, Berkeley, Calif. 94720, USA
WILMA
Dissociation of embryos of U. caupo prior to the blastula stage does not retard the apparent initiation of ribosomal ribonucleic acid (rRNA). The state of aggregation of the dissociated embryos does not seem-to -influence the initiation or continuation of synthesis of rRNA. It is concluded that cell interaction is not necessary for commitment to continuing rRNA synthesis.
Materials and Methods Collection and culture of gametes [9] and removal of the fertilization membrane (FM) have been described [7]. In most experiments, PSSW (0.1 mg each of uenicillin and streotomvciniml Millinore-filtered sea water) at 15°C was used though no differences were detected when antibiotics were absent. .
.
Dissociation: Membraneless embryos were washed with calcium-free sea water and 1 mM disodium ethylenediamine tetraacetate (EDTA), pH 7.8, centrifuged and resuspended in 15 vol of 0.44 M sucrose, 1 mM EDTA, and 0.01 M Tris, pH 8.0 [15] at 15°C; pipetted gently until single cells, and diluted threefold witkPSSW. The suspension of single cells was centrifuged at 2 000 rpm, 1) min in a clinical centrifuge; washed in PSSW, and resuspended so that a ‘monolayer’ of cells was obtained in culture dishes. Cultures of dissociated embryos were gently shaken after 10 and 60 min because cells adhering to the siliconized substrate did not undergo normal development.
A change in the relative rate of synthesis of rRNA is detected at or shortly after gastruIncubation in isotope: Unfertilized eggs were prelation in many vertebrate [I, 31 and invertelabeled 1 to 14 h with 2 /Xi 8H-nucleoside(s) each/ml brate [4, 181 embryos. This ‘initiation’ of then twice centrifuged, resuspended and fertilized as rRNA synthesis might be influenced by new previously described. specific cell associations. Alternatively, there Extraction of RNA: Embryos were washed in cold Millioore-filtered sea water, centrifuged and frozen. may be a necessity for a certain time period Frozen pellets were homogenized at2”C with 20-25 of association between blastomeres rather vol of 0.1 M acetate buffer (pH 5), 10 pg polyvinylsulfonate (PVS)/ml, bentonite (1 mg/ml) and 2% than specific associations of the gastrula stage sodium dodecvlsulfate (SDS). RNA was extracted per se. with phenol, 6OC [22], ‘and ‘precipitated in ethanol. The pellet was dissolved in DNase buffer ((0.01 M AC The approach was to dissociate embryos buffer (pH 5), 1 mM MgCl,, 10 pg PVS/ml)) and into individual blastomeres at specific times incubated with 15 ua DNase/ml. 30 min (18-20°C). Some samples received 50 pg’prdnase (self:digested)/ and observe whether they synthesized rRNA ml for 60 min (20°C). The chilled preparation was concurrently with the intact controls. Urechis re-extracted with phenol (4°C) and 1 % SDS. Prior to ethanol precipitation, the aqueous phase was dialysed eggs undergo spiral determinate cleavage in 0.1 M AC buffer containing 1 mM EDTA and 10 pg characteristic of ‘mosaic’ eggs. PVS/ml. The final RNA pellet was dissolved in 0.01 M AC buffer containing 1 mM EDTA. The present results show that initiation Sucrose gradient centrifugation: Gradients were and/or continuation of rRNA synthesis is 5-20 % sucrose (0.1 M AC buffer, pH 5,l mM EDTA). not influenced by normal cell associations, Fractions received protein carrier, were precipitated with cold 5 % tetrachloroacetic acid (TCA) onto glass a conclusion similar to the interpretations fiber filters and counted in toluene-scintillation fluid. reached in other experiments with so-called In selected experiments, filters were removed from vials representing a 28s peak and incubated in 0.3 regulative embryos [16, 20, 211. r Present address: Department of Biology, University of Oregon, Eugene, Oreg. 97403, USA. Exptl Cell Res 72
N KOH at 37°C for 24 h. The alkaline-hydrolysable and acid-precipitable counts in the KOH mixture were determined, the former averaged 98.7 % (range 96.0 to 100%).
RNA synthesis in dissociated embryos
0.25 0.20 0.15.
O.lO200
0.05.
0 5
IO
1'5
bottom
20
539
Figs 1-2. Abscissa: fraction number; ordinafe: (left) A& (right) cpm. Fig. 1. RNA synthesis in embryos prelabeled with 8H-5-uridine (20-25 Ci/mM) as described under Methods. Unless otherwise noted, all gradients were centrifuged in the Spinco SW39 rotor at 39 000 rpm for 3t h. Absorbancies were read at 260 nm and were assumed to be 28, 18 and 4S [lo]. O---O, absorbance; cpm at A, 17 h; o, 18% h; n, 20 h; q , 77 h.
25 top
Isotope uptake and incorporation into RNA: These were determined by perchloric acid extractions and base hydrolysis using modified methods of SchmidtThannhauser [17]. Extracts dried on glass fiber filters were counted as described above. Autoradiography: Embryos were incubated with 50 PCi SH-uridine/ml, washed, fixed in acetic acid-alcohol (1: 3) at 2°C (2-18 h) and paraffin-embedded. Sections were incubated with DNase (0.17 ma/ml of DNase buffer) and control sections with RNase (preheated 10 min at 90°C) at 37°C for 34 h. Washed sections were placed in cold 3.5 % PCA. Slides were dipped in Ilford L-5 emulsion, exposed for about 14 days and developed in D-19.
Results Synthesis of ribosomal RNA
Only the 28s region of sucrose gradients was evaluated, since Gould [lo] and the present investigator found heterodisperse RNA in the 10-20s region is synthesized before the blastula stage in Urechis. It is likely that dissociation might affect permeability and pool sizes; thus embryos were prelabeled with 3Hnucleosides prior to dissociation. Pulse labeling studies with methyl labeled methionine were not as sensitive as nucleoside prelabeling, nor were the results with methionine pulses at variance in any way with the conclusions drawn here (unpublished). Embryos were prelabeled with 3H-uridine for 1 h prior to fertilization (data not presented here showed that an excess of tritium was present in the acid soluble fraction). From 17 h (early gastrula) to 20 h (mid-gastrula), fig. 1, the patterns of tritium in the 28s
region progressed from the occurrence of a shoulder of radioactivity to a clearly detectable peak coincident with the 28 S absorbance peak. The radioactivity pattern in 20 h-old embryos is like that seen by Gould [lo] at 162 h in Urechis embryos grown at 17°C. The specific activities of the 28s region progressively increased with development of the trochophore larva, 423 and 77 h (fig. 1). Extracts of RNA from ‘membraneless’ embryos gave comparable patterns of radioactivity, with a definite peak of radioactivity at 21 h even though there were minor differences in their morphology [7]. It was concluded that a detectable level of synthesis of rRNA was initiated in embryos by at least 21 h (mid-gastrula stage) of development and that accumulation of rRNA continued for at least 57 h. Removal of the FM did not appreciably retard the initiation of synthesis of rRNA. Effect of dissociation of embryos upon rRNA synthesis
It was of interest to determine if the intact embryos and/or the morphogenetic movements of gastrulation are necessary for the expression of the initiation of detectable rRNA synthesis. Prelabelled membraneless embryos were dissociated after 16 h of development, shortly before gastrulation. The patterns of radioacExptl Cell Res 72
540
Wilma Engstrom
200 cells. Thus, it can be seen that even a suspension of single cells is able to maintain rRNA synthesis. Autoradiography
Fig. 2. RNA synthesis in q , 17 h and 0, 21 h embryos dissociated at 16 h post-fertilization; A, 21% h embryos dissociated at 7 h post-fertilization; A, 21 h control embryos. Embryos are from eggs prelabeled with q , 3H-uridine only, or o and A, SH-uridine with sH-8quanosine (26.8 Ci/mM), or A, SH-cytidine (5.06 Ci/mM) as described in fig. 1. Gradients centrifuged for 44 h. O-O, absorbance.
tivity of the isolated cells of 17 and 21 h embryos were like those of the controls (fig. 2). One can conclude that neither an intact embryo nor the movements of gastrulation are required for accumulation of detectable rRNA during the gastrula stage. The possibility that there is a critical period of time during which cells must be in contact for commitment to synthesis to occur was investigated by dissociating embryos at earlier times, 7 h postfertilization (40+ to 56 cells) or 5 h 50 min postfertilization (about 32 cells), and harvesting at 21 h. It can be seen from fig. 2 that dissociation as early as 7 h of development did not retard the initiation of detectable synthesis of rRNA. The 28 S peak of radioactivity was not as well defined in the earlier dissociated sample. Just as in control embryos, continued culture (up to 77 h) of the dissociated embryos (from all dissociation times) lead to an increase in the specific activity of the 28 S region. The rate and extent of aggregation of cells varied between experiments as well as between duplicate cultures, ranging from mostly single cells to small motile clumps to large loose clusters of about Exptl
Cell Res 72
Autoradiograms were prepared from FM + , FM - , and dissociated embryos labeled continuously from 5 h after fertilization to the time of harvesting. By 64 h (24-40 cells) the nucleolus was synthetically active. Embryos dissociated at 5 h gave the same autoradiographic picture as controls at 7& h (data were not collected at 6Q h). Grains were RNase sensitive at all stages. Gould [lo] also found grains over the nucleolus prior to the swimming blastula stage. This contrasts with other embryos in which no RNA synthesis is detected in the nucleolus until gastrulation [2, 121. The inability to detect rRNA synthesis prior to the 20 h gastrula stage by sucrose gradient analysis even though the nucleoli were synthetically active could be a reflection of synthesis of rRNA precursor with little or no conversion to 28S, synthesis of rRNA but resistent to phenol extraction or synthesized in amounts too low to be detected in the presence of heterodisperse RNA (cf [6]), or synthesis or sequestration by the nucleolus of RNA other than rRNA. Discussion
While this work was in progress, it was reported that dissociated Xenopus Iaevis midblastulae or late neurulae [ 16, 211 or mid-gastrulae [ 141 and dissociated Paracentrotus Zividusmid-gastrulae [8] initiated rRNA synthesis at the same time as the undissociated control embryos and that dissociated hatching blastulae of P. lividus did not display rRNA synthesis at the appropriate time [8]. More recently evidence was presented that dissociated hatching blastulae may, indeed, be capable of normal rRNA synthesis [20]. In the present study, dissociation of em-
On the nuclear position in onion root-tip cells
541
4. Comb, D G, Katz, S, Branda, R & Pinzino, C, bryos two and perhaps three hours prior to J mol biol 14 (1965) 195. the blastula stage or at the beginning of 5. Das, N K, Luykx, P & Alfert, M, Dev biol 12 (1965) 72. gastrulation did not seem to retard apparent 6. Emerson, C & Humphreys, T, Dev biol 23 initiation of synthesis of rRNA. Studies of (1970) 86. 7. Engstrom, W, Biol bull 140 (1971) 369. amphibia [14, 16, 211 and sea urchins [20] 8. Giudice, G, Mutolo, V & Moscona, A, Biochim and the present data from ‘mosaic’ Urechis biophys acta 138 (1967) 607. 9. Gould, M, Methods in developmental biology embryos all support the conclusion that cell (ed F Wilt & N Wessels) p. 163. Crowell, New dissociation does not interfere with the York (1967). 10. Gould, M, Dev biol 19 (1969) 482. appearance of detectable rRNA synthesis. In 11. Hay, E D & Gurdon, J B, J cell sci 2 (1967) 151. evaluating the synthesis of rRNA in Urechis 12. Karasaki, S, Exptl cell res 52 (1968) 13. embryos, it was assumed that coincidence of 13. Kaulenas, M, Foor, W & Fairbairn, D, Science 163 (1969) 1201. radioactivity and absorbance peaks in the 14. Landesman, R & Gross, P, Dev biol 18 (1968) 571. 28s region by 21 h represented rRNA [lo] 15. Millonig, G & Giudice, G, Dev biol 15 (1967) and reflected the in vivo situation. It is likely 91. (cf [6]) that lower levels of rRNA synthesis 16. Miyahara, E, Shiokawa, K & Yamana, K, Proc jap acad 44 (1968) 697. occurred in earlier stages but were undetected 17. Munro, H & Fleck, A, Methods biochem anal 14 (1966) 113. by sucrose gradient analysis. 18. Nemer, M, Proc natl acad sci US 50 (1963) 230. Perhaps the nucleolus itself has a role in 19. Schwartz, M, Carnegie year book (1967-68) 413. the regulation of rRNA synthesis. Cleavage 20. Sconzo, G, Pirrone, A, Mutolo, V & Giudice, G, Biochim biophys acta 199 (1970) 441. stages of X. Iaevis and Arbacia punctulata 21. Shiokawa, K & Yamana, K, Dev biol 16 (1967) 368. contain prenucleolar bodies which become 22. Slater, D, & Spiegelman, S, Biophys j 6 (1966) definitive nucleoli in the gastrula stage [l 1, 386. 121. It is not known whether prenucleolar bodies exist in the later cleavage stages of Received May 27, 1971 Revised version received February 7, 1972 Urechis; the nucleolus has disappeared by the end of the first meiotic division [5]. No rRNA synthesis has been detected until the definitive nucleolus stage in other organisms [2, 12, 131. Effects of colchiciae aad high hydrostatic pressure on the nuclear In the present autoradiographic study, RNA position in onion root-tip cells synthesis in the nucleolus was demonstrated three hours prior to the swimming blastula R. MOLINAl and J. F. LOPEZ-SAEZ, Department stage, i.e., between 24 and 40 cells, even of Morphology and Physiology, CSIC, Faculty of Sciences, Seville, Spain though synthesis could not be detected by sucrose gradient analysis or MAK column chromatography [19] until the gastrula stage. The root-tip cell possesses a mechanism by which the My sincere gratitude to Fred Wilt for thoughtful and stimulating discussions and guidance. This work was supported in part by grant GM 13882 from USPHS to Fred H. Wilt and a National Institutes of Health predoctoral fellowship to W.E.
References 1. Brown, 2. Brown, sci US 3. Brown,
D & Caston, J, Dev biol 5 (1962) 435. D D & Gurdon, J, B, Proc‘natl’ acad 51 (1964) 139. D & Littna, E, J mol biol 8 (1964) 669.
position of the nucleus is actively controlled. Our preliminary observations demonstrated that the cell is capable of restoring the nucleus to its original position when it has been displaced by centrifugal force. The aim of the present study is to approach the problem of the nuclear position in meristematic cells by examining its normal position in cells from cut roots, its displacement in centrifuged roots, and the return to normal position in the course of recovery with or without various treatments. Immediately after centri1 Address: Catedra de Citologla, Facultad de Ciencias, C/. Palos de la Frontera s/n, Sevilla, Spain. Exptl Cell Res 72
NOTES
Effect of dissociation of embryos of Urechis caupo on synthesis of ribosomal RNA ENGSTROM,’ Department of Zoology, University of California, Berkeley, Calif. 94720, USA
WILMA
Dissociation of embryos of U. caupo prior to the blastula stage does not retard the apparent initiation of ribosomal ribonucleic acid (rRNA). The state of aggregation of the dissociated embryos does not seem-to -influence the initiation or continuation of synthesis of rRNA. It is concluded that cell interaction is not necessary for commitment to continuing rRNA synthesis.
Materials and Methods Collection and culture of gametes [9] and removal of the fertilization membrane (FM) have been described [7]. In most experiments, PSSW (0.1 mg each of uenicillin and streotomvciniml Millinore-filtered sea water) at 15°C was used though no differences were detected when antibiotics were absent. .
.
Dissociation: Membraneless embryos were washed with calcium-free sea water and 1 mM disodium ethylenediamine tetraacetate (EDTA), pH 7.8, centrifuged and resuspended in 15 vol of 0.44 M sucrose, 1 mM EDTA, and 0.01 M Tris, pH 8.0 [15] at 15°C; pipetted gently until single cells, and diluted threefold witkPSSW. The suspension of single cells was centrifuged at 2 000 rpm, 1) min in a clinical centrifuge; washed in PSSW, and resuspended so that a ‘monolayer’ of cells was obtained in culture dishes. Cultures of dissociated embryos were gently shaken after 10 and 60 min because cells adhering to the siliconized substrate did not undergo normal development.
A change in the relative rate of synthesis of rRNA is detected at or shortly after gastruIncubation in isotope: Unfertilized eggs were prelation in many vertebrate [I, 31 and invertelabeled 1 to 14 h with 2 /Xi 8H-nucleoside(s) each/ml brate [4, 181 embryos. This ‘initiation’ of then twice centrifuged, resuspended and fertilized as rRNA synthesis might be influenced by new previously described. specific cell associations. Alternatively, there Extraction of RNA: Embryos were washed in cold Millioore-filtered sea water, centrifuged and frozen. may be a necessity for a certain time period Frozen pellets were homogenized at2”C with 20-25 of association between blastomeres rather vol of 0.1 M acetate buffer (pH 5), 10 pg polyvinylsulfonate (PVS)/ml, bentonite (1 mg/ml) and 2% than specific associations of the gastrula stage sodium dodecvlsulfate (SDS). RNA was extracted per se. with phenol, 6OC [22], ‘and ‘precipitated in ethanol. The pellet was dissolved in DNase buffer ((0.01 M AC The approach was to dissociate embryos buffer (pH 5), 1 mM MgCl,, 10 pg PVS/ml)) and into individual blastomeres at specific times incubated with 15 ua DNase/ml. 30 min (18-20°C). Some samples received 50 pg’prdnase (self:digested)/ and observe whether they synthesized rRNA ml for 60 min (20°C). The chilled preparation was concurrently with the intact controls. Urechis re-extracted with phenol (4°C) and 1 % SDS. Prior to ethanol precipitation, the aqueous phase was dialysed eggs undergo spiral determinate cleavage in 0.1 M AC buffer containing 1 mM EDTA and 10 pg characteristic of ‘mosaic’ eggs. PVS/ml. The final RNA pellet was dissolved in 0.01 M AC buffer containing 1 mM EDTA. The present results show that initiation Sucrose gradient centrifugation: Gradients were and/or continuation of rRNA synthesis is 5-20 % sucrose (0.1 M AC buffer, pH 5,l mM EDTA). not influenced by normal cell associations, Fractions received protein carrier, were precipitated with cold 5 % tetrachloroacetic acid (TCA) onto glass a conclusion similar to the interpretations fiber filters and counted in toluene-scintillation fluid. reached in other experiments with so-called In selected experiments, filters were removed from vials representing a 28s peak and incubated in 0.3 regulative embryos [16, 20, 211. r Present address: Department of Biology, University of Oregon, Eugene, Oreg. 97403, USA. Exptl Cell Res 72
N KOH at 37°C for 24 h. The alkaline-hydrolysable and acid-precipitable counts in the KOH mixture were determined, the former averaged 98.7 % (range 96.0 to 100%).
RNA synthesis in dissociated embryos
0.25 0.20 0.15.
O.lO200
0.05.
0 5
IO
1'5
bottom
20
539
Figs 1-2. Abscissa: fraction number; ordinafe: (left) A& (right) cpm. Fig. 1. RNA synthesis in embryos prelabeled with 8H-5-uridine (20-25 Ci/mM) as described under Methods. Unless otherwise noted, all gradients were centrifuged in the Spinco SW39 rotor at 39 000 rpm for 3t h. Absorbancies were read at 260 nm and were assumed to be 28, 18 and 4S [lo]. O---O, absorbance; cpm at A, 17 h; o, 18% h; n, 20 h; q , 77 h.
25 top
Isotope uptake and incorporation into RNA: These were determined by perchloric acid extractions and base hydrolysis using modified methods of SchmidtThannhauser [17]. Extracts dried on glass fiber filters were counted as described above. Autoradiography: Embryos were incubated with 50 PCi SH-uridine/ml, washed, fixed in acetic acid-alcohol (1: 3) at 2°C (2-18 h) and paraffin-embedded. Sections were incubated with DNase (0.17 ma/ml of DNase buffer) and control sections with RNase (preheated 10 min at 90°C) at 37°C for 34 h. Washed sections were placed in cold 3.5 % PCA. Slides were dipped in Ilford L-5 emulsion, exposed for about 14 days and developed in D-19.
Results Synthesis of ribosomal RNA
Only the 28s region of sucrose gradients was evaluated, since Gould [lo] and the present investigator found heterodisperse RNA in the 10-20s region is synthesized before the blastula stage in Urechis. It is likely that dissociation might affect permeability and pool sizes; thus embryos were prelabeled with 3Hnucleosides prior to dissociation. Pulse labeling studies with methyl labeled methionine were not as sensitive as nucleoside prelabeling, nor were the results with methionine pulses at variance in any way with the conclusions drawn here (unpublished). Embryos were prelabeled with 3H-uridine for 1 h prior to fertilization (data not presented here showed that an excess of tritium was present in the acid soluble fraction). From 17 h (early gastrula) to 20 h (mid-gastrula), fig. 1, the patterns of tritium in the 28s
region progressed from the occurrence of a shoulder of radioactivity to a clearly detectable peak coincident with the 28 S absorbance peak. The radioactivity pattern in 20 h-old embryos is like that seen by Gould [lo] at 162 h in Urechis embryos grown at 17°C. The specific activities of the 28s region progressively increased with development of the trochophore larva, 423 and 77 h (fig. 1). Extracts of RNA from ‘membraneless’ embryos gave comparable patterns of radioactivity, with a definite peak of radioactivity at 21 h even though there were minor differences in their morphology [7]. It was concluded that a detectable level of synthesis of rRNA was initiated in embryos by at least 21 h (mid-gastrula stage) of development and that accumulation of rRNA continued for at least 57 h. Removal of the FM did not appreciably retard the initiation of synthesis of rRNA. Effect of dissociation of embryos upon rRNA synthesis
It was of interest to determine if the intact embryos and/or the morphogenetic movements of gastrulation are necessary for the expression of the initiation of detectable rRNA synthesis. Prelabelled membraneless embryos were dissociated after 16 h of development, shortly before gastrulation. The patterns of radioacExptl Cell Res 72
540
Wilma Engstrom
200 cells. Thus, it can be seen that even a suspension of single cells is able to maintain rRNA synthesis. Autoradiography
Fig. 2. RNA synthesis in q , 17 h and 0, 21 h embryos dissociated at 16 h post-fertilization; A, 21% h embryos dissociated at 7 h post-fertilization; A, 21 h control embryos. Embryos are from eggs prelabeled with q , 3H-uridine only, or o and A, SH-uridine with sH-8quanosine (26.8 Ci/mM), or A, SH-cytidine (5.06 Ci/mM) as described in fig. 1. Gradients centrifuged for 44 h. O-O, absorbance.
tivity of the isolated cells of 17 and 21 h embryos were like those of the controls (fig. 2). One can conclude that neither an intact embryo nor the movements of gastrulation are required for accumulation of detectable rRNA during the gastrula stage. The possibility that there is a critical period of time during which cells must be in contact for commitment to synthesis to occur was investigated by dissociating embryos at earlier times, 7 h postfertilization (40+ to 56 cells) or 5 h 50 min postfertilization (about 32 cells), and harvesting at 21 h. It can be seen from fig. 2 that dissociation as early as 7 h of development did not retard the initiation of detectable synthesis of rRNA. The 28 S peak of radioactivity was not as well defined in the earlier dissociated sample. Just as in control embryos, continued culture (up to 77 h) of the dissociated embryos (from all dissociation times) lead to an increase in the specific activity of the 28 S region. The rate and extent of aggregation of cells varied between experiments as well as between duplicate cultures, ranging from mostly single cells to small motile clumps to large loose clusters of about Exptl
Cell Res 72
Autoradiograms were prepared from FM + , FM - , and dissociated embryos labeled continuously from 5 h after fertilization to the time of harvesting. By 64 h (24-40 cells) the nucleolus was synthetically active. Embryos dissociated at 5 h gave the same autoradiographic picture as controls at 7& h (data were not collected at 6Q h). Grains were RNase sensitive at all stages. Gould [lo] also found grains over the nucleolus prior to the swimming blastula stage. This contrasts with other embryos in which no RNA synthesis is detected in the nucleolus until gastrulation [2, 121. The inability to detect rRNA synthesis prior to the 20 h gastrula stage by sucrose gradient analysis even though the nucleoli were synthetically active could be a reflection of synthesis of rRNA precursor with little or no conversion to 28S, synthesis of rRNA but resistent to phenol extraction or synthesized in amounts too low to be detected in the presence of heterodisperse RNA (cf [6]), or synthesis or sequestration by the nucleolus of RNA other than rRNA. Discussion
While this work was in progress, it was reported that dissociated Xenopus Iaevis midblastulae or late neurulae [ 16, 211 or mid-gastrulae [ 141 and dissociated Paracentrotus Zividusmid-gastrulae [8] initiated rRNA synthesis at the same time as the undissociated control embryos and that dissociated hatching blastulae of P. lividus did not display rRNA synthesis at the appropriate time [8]. More recently evidence was presented that dissociated hatching blastulae may, indeed, be capable of normal rRNA synthesis [20]. In the present study, dissociation of em-
On the nuclear position in onion root-tip cells
541
4. Comb, D G, Katz, S, Branda, R & Pinzino, C, bryos two and perhaps three hours prior to J mol biol 14 (1965) 195. the blastula stage or at the beginning of 5. Das, N K, Luykx, P & Alfert, M, Dev biol 12 (1965) 72. gastrulation did not seem to retard apparent 6. Emerson, C & Humphreys, T, Dev biol 23 initiation of synthesis of rRNA. Studies of (1970) 86. 7. Engstrom, W, Biol bull 140 (1971) 369. amphibia [14, 16, 211 and sea urchins [20] 8. Giudice, G, Mutolo, V & Moscona, A, Biochim and the present data from ‘mosaic’ Urechis biophys acta 138 (1967) 607. 9. Gould, M, Methods in developmental biology embryos all support the conclusion that cell (ed F Wilt & N Wessels) p. 163. Crowell, New dissociation does not interfere with the York (1967). 10. Gould, M, Dev biol 19 (1969) 482. appearance of detectable rRNA synthesis. In 11. Hay, E D & Gurdon, J B, J cell sci 2 (1967) 151. evaluating the synthesis of rRNA in Urechis 12. Karasaki, S, Exptl cell res 52 (1968) 13. embryos, it was assumed that coincidence of 13. Kaulenas, M, Foor, W & Fairbairn, D, Science 163 (1969) 1201. radioactivity and absorbance peaks in the 14. Landesman, R & Gross, P, Dev biol 18 (1968) 571. 28s region by 21 h represented rRNA [lo] 15. Millonig, G & Giudice, G, Dev biol 15 (1967) and reflected the in vivo situation. It is likely 91. (cf [6]) that lower levels of rRNA synthesis 16. Miyahara, E, Shiokawa, K & Yamana, K, Proc jap acad 44 (1968) 697. occurred in earlier stages but were undetected 17. Munro, H & Fleck, A, Methods biochem anal 14 (1966) 113. by sucrose gradient analysis. 18. Nemer, M, Proc natl acad sci US 50 (1963) 230. Perhaps the nucleolus itself has a role in 19. Schwartz, M, Carnegie year book (1967-68) 413. the regulation of rRNA synthesis. Cleavage 20. Sconzo, G, Pirrone, A, Mutolo, V & Giudice, G, Biochim biophys acta 199 (1970) 441. stages of X. Iaevis and Arbacia punctulata 21. Shiokawa, K & Yamana, K, Dev biol 16 (1967) 368. contain prenucleolar bodies which become 22. Slater, D, & Spiegelman, S, Biophys j 6 (1966) definitive nucleoli in the gastrula stage [l 1, 386. 121. It is not known whether prenucleolar bodies exist in the later cleavage stages of Received May 27, 1971 Revised version received February 7, 1972 Urechis; the nucleolus has disappeared by the end of the first meiotic division [5]. No rRNA synthesis has been detected until the definitive nucleolus stage in other organisms [2, 12, 131. Effects of colchiciae aad high hydrostatic pressure on the nuclear In the present autoradiographic study, RNA position in onion root-tip cells synthesis in the nucleolus was demonstrated three hours prior to the swimming blastula R. MOLINAl and J. F. LOPEZ-SAEZ, Department stage, i.e., between 24 and 40 cells, even of Morphology and Physiology, CSIC, Faculty of Sciences, Seville, Spain though synthesis could not be detected by sucrose gradient analysis or MAK column chromatography [19] until the gastrula stage. The root-tip cell possesses a mechanism by which the My sincere gratitude to Fred Wilt for thoughtful and stimulating discussions and guidance. This work was supported in part by grant GM 13882 from USPHS to Fred H. Wilt and a National Institutes of Health predoctoral fellowship to W.E.
References 1. Brown, 2. Brown, sci US 3. Brown,
D & Caston, J, Dev biol 5 (1962) 435. D D & Gurdon, J, B, Proc‘natl’ acad 51 (1964) 139. D & Littna, E, J mol biol 8 (1964) 669.
position of the nucleus is actively controlled. Our preliminary observations demonstrated that the cell is capable of restoring the nucleus to its original position when it has been displaced by centrifugal force. The aim of the present study is to approach the problem of the nuclear position in meristematic cells by examining its normal position in cells from cut roots, its displacement in centrifuged roots, and the return to normal position in the course of recovery with or without various treatments. Immediately after centri1 Address: Catedra de Citologla, Facultad de Ciencias, C/. Palos de la Frontera s/n, Sevilla, Spain. Exptl Cell Res 72