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RNA synthesis in roots of Vicia faba
Experimentul
Cell Research
35, 317-325
(1964)
RNA SYNTHESIS
317
IN ROOTS OF VlCIA
FABA
D. DAVIDSON1 Biology
Division,
Oak
Ridge
National Received
Laboratory,’ July
Oak Ridge,
Term.,
U.S.A.
25, 1963
SYKTIIESIS of RNA has now been studied in a number of plant and animal cells by using radioactive precursors and autoradiography combined with such different experimental approaches as nuclear transplantation [a] and enucleation [5, 15-171. The published evidence [15, 18, 19, 21-23, 26-281 strongly supports two general conclusions: (a) RNA is synthesized in the nucleus, and (b) RNA moves from the nucleus to the cytoplasm. In some cells the site of most active synthesis within the nucleus is the nucleolus [15, 261, cf., however [3, 231. The incorporation of labeled precursors does not occur during the stages of mitosis [20, 23] when the nucleus is no longer an intact organelle. RNA has been described as a constituent of the mitotic chromosome on the basis of results from double-staining treatments combined with exposure to specific enzymes (see discussions [l, 91). The results reported here do not support the conclusion that RNA present in the prophase nucleus becomes a constituent of the prophase and metaphase chromosome; consequently, they preclude the possibility of loss of RNA from chromosomes during anaphase movement [cf. 7, 8, 11, la]. The present study was undertaken, first, to determine how the incorporation of isotopically labeled precursors into cells of I’icia roots \\-as affected by the onset of mitosis, and secondly, to estimate the fate of nuclear RKA during prophase and to determine the contribution of RNA’ to the composition of the mitotic chromosome. Evidence is presented that RNA synthesis occurs only in the nucleus and not in the cytoplasm; alsb that synthesis of RNA in the nucleus ceases suddenly toward the end of prophase and does not occw while the chromosomes are in the condensed f&m. The chromosomes appear to be capable of synthesis only when they are in the dispersed condition of interphase or in the partially condensed stage of early prophase xvhile they are contained within an intact nuclear membrane. 1 f’resent Scotland. * Operated
address: by Union
Department Carbide
of Botany, Corporation
University for the United
of St. States
Andrew, Atomic
St. Energy
Experimental
Andrew,,
Fife,
Commission. Cell Research
35
318
D. Davidson MATERIAL
AND
METHODS
Beans (Vicia f&a) were germinated and grown in aerated distilled water until lateral roots appeared. The root system was suspended in solutions of 3H-cytidine or 3H-uridine for from 30 min to 2 hr. The activity of both labeled solutions was 2 PC/ml. Immediately after exposure roots \vere fixed in chilled acetic acid-alcohol (1 : 3) overnight. They were washed and then softened by immersion for 24 hr in a 1 per cent (w/v) solution of pectinase (pH 3.9). This treatment dissolves the middle lamella between cells allowing the cells to separate when pressed very gently, The apical meristems of roots were later removed and prepared as squashes. Cold TCA (5 per cent) was used to remove acid-soluble RNA but it does not soften the cells. Good preparations with well-flattened cells were obtained by using 45 per cent (v/v) acetic acid, which softens the cells. Control roots, also exposed to 3H-cytidine or 3H-uridine were prepared as squashes and incubated in buffered RNAase (1 mg/5 cc) at room temperature for 1 hr. After this treatment, grain counts over cells were almost the same as background counts, indicating that almost all radioactive material had been removed. Slides were frozen on dr>- ice before removal of coverslips with a razor blade. Squashes were coated with a liquid emulsion (Kodak, NTB2) and exposed for 2 weeks. After photographic processing, preparations were stained in toluidine blue, dehydrated, and mounted in I-uparal. In making grain counts, correction was made for background grains by counting grains in an adjacent area equal to the area of the cell. RESULTS
Exposure for 30 min and 1 hr.-Roots were exposed to solutions of 3Hcytidine and 3H-uridine for 30 min and 1 hr. In scoring preparations of roots grown in 3H-cytidine for 1 hr, the number of grains over nuclei of different sizes was counted. Incorporation of 3H-cytidine occurred in nuclei of all sizes. If nuclear size is taken as an indicator of degree of development within the cell cycle, it appears that RNA synthesis is continuous throughout interphase. In roots exposed to 3H-cytidine for 1 hr, mean grain counts over nuclei of different sizes (average of 50 cells) were: 13.1 + 1.5 (95 per cent confidence limits) in cells with largest nuclei, 6.7 + 1.l in cells with smallest nuclei, and 14.5 * 1.02 for cells of all sizes of nuclei. The total amount of incorporation of labeled precursor was similar in both 30-min exposures (Tables 1 and 11) but was unaccountably higher when 3H-uridine rather than 3H-cytidine was supplied to the roots for 1 hr (cf. Tables Ill and IV). Apart from this difference in the actual numbers of grains per cell, the direction of change in grain counts with time or between different stages of the mitotic cycle, were similar with both compounds. After a 30-min treatment with 3H-cytidine (Table 1) or uridine (Table II), grain counts were low over cells in metaphase, anaphase, and telophase. Experimenlnl
Cell
Research
35
RNA
synthesis
in roots of Vicia
faba
It is evident that incorporation of labeled precursors is reduced in cells undergoing mitosis. In such cells, more grains occur over the cytoplasm than over the chromosomes. In cells at early prophase and interphase, from the same roots, however, the majority of grains are over the nuclei. The relative distribution of grains is given by the ratio of grains over the nucleus (or chromosomes in dividing cells) to grains over the cytoplasm. This ratio is higher in prophase and interphase cells than in dividing cells (Tables I-IV). Later it will be shown that the presence of grains over the cytoplasm of cells in mitosis is not the result of cytoplasmic synthesis of RNA; nuclear synthesis of labeled RNA in the previous prophase or interphase, followed by movement to the cytoplasm, can account for grains over the cytoplasm at metaphase. TABLE
1. Grain counts in cells of roots of V. faba exposed to 3H-cytidine for 30 minutes.
Stage Interphase Prophase Metaphase Anaphase Telophase a Mean confidence to zero.
TABLE
Total no. grains 834 282 165 61 8
no. grains per cella 8.3i- 1.1 5.6Fl.l 3.3 Al 0.9 1.2 -
number of grains per cell is given limits are given for the measurements
II.
Mean
Ratio nucleus/ cytoplasm
Total cells
3.3/l 2.711 0.4/l 0.4/l
with 95 per cent confidence of anaphase and telophase
100 50 50 50 50 limits. because
No measure of the means tend
Grain counts in cells of roots of V. faba exposed to 3H-uridine for 30 minutes.
Stage Prophase Early Middle Late Metaphase Anaphase Telophase a See footnote
Total no. grains
162 164 39 33 17 23 ‘, Table
Mean
no. grains per cella
8.lk 2.2 8.2 It 1.7 2.0 k 0.5 3.3 i 1.9 1.7 2.3
Ratio nucleus/ cytoplasm
Total cells
3.8/l 1.4/l 0.411 0.3/l 0.5/l 0.5/l
20 20 20 10 10 10
I. Ex,perimenlal
Cell Research
35
320
D. Davidson
Active incorporation of the labeled compounds occurred in prophase c.ells (Tables I-V). I n cells treated with 3H-cytidine for either 30 min or 1 hr (Tables I and III) a fall in the ratio of nuclear to cytoplasmic grain counts from 1 hr to 30 min (i.e., from interphase through the various stages of mitosis) was found; this was the first evidence of a marked loss of nuclear RNA to the cytoplasm during prophase. Prophase cells of roots that had been grown in 3H-uridine were classified as early, middle, and late prophase on the degree of chromosome contraction. It is clear that incorporation occurs in early and middle prophase but is greater in early prophase (Tables II and IV). In both treatments it is also evident that in late prophase labeled RNA moves from the nucleus to the cytoplasm, giving a changed nucleus/ cytoplasmic ratio. Thus the ratio of grain counts is 3.8/l in early prophase and 0.4/l in late prophase in cells treated with 3H-uridine for 30 min (Table II). TABLE
III.
Grain counts in cells of roots of V. faba exposed to 3H-cytidine for 1 hour. Total no. grains
Stage Interphase Prophase Metaphase Anaphase Telophase a See footnote T.~BI,E
Mean
14.5Fl.O 14.12 1.8 7.3 i 2.0 5.4k3.0 2.3 i: 1.4
2175 705 365 270 115 ‘, Table
no. grains per cella
Ratio nucleus/ cytoplasm
Total cells
2.9/l 1.2/l 0.5/l 0.5/l 0.3/l
150 50 50 50 50
I.
IV. Grain counts in cells of roots of V. faba exposed to 3H-uridine Total no. grains
Stage
no. grains per cella
Ratio nucleus/ cytoplasm
Total cells
Interphase Prophase
4193
41.9 f 6.5
1.5/l
100
Early Middle Late Metaphase Anaphase Telophase
932 903 892 844 671 430
‘16.6 + 1.4 45.2 k 6.2 44.6 IL 5.3 33.8 -t 3.5 26.8 + 3.4 17.2ri-1.9
2.0/l 1.3/l 0.7/l 0.5/l 0.6/l 0.3/l
20 20 20 25 25 25
a See footnote Experimental
lIean
for 1 hour.
Cell Research
‘, Table 35
I.
RNA
synthesis
in roots of Vicia
faba
This change of ratio and the failure to find large numbers of grains over metaphase chromosomes indicates that (a) there is little synthesis of RNA in late prophase, and (b) most of the RNA present in the nucleus moves to the cytoplasm and very little of it becomes a constituent of the metaphase chromosomes. TARLE V. Grain counts in cells of roots of V. faba exposed to 3H-uridine
Stage Interphase Prophase Metaphase Anaphase Telophase a See footnote
Total no. grains
10,810 5342 3757 3355 2592 a, Table
Mean
no. grains per cella
108.1? 106.8 75.1 67.1 51.8
k i k k
7.5 7.7 7.8 8.1 6.S
Ratio nucleus/ cytoplasm
1.4/l 1.3/l 0.7/l 0.8/l 0.3/l
for 2 hour.
Total cells
100 50 50 50 50
I.
Doubling the duration of treatments resulted in a marked increase, almost a doubling, in the mean number of grains per interphase cell (Tables I-IV). The nucleus-to-cytoplasm ratio of the mean number of grains also changes with time. It decreases slightly as the actual mean number of grains per cell increases. This change in the pattern of the distribution of labeled RNA in interphase cells appears to be in agreement with the evidence that RNA is synthesized in the nucleus and moves to the cytoplasm. Further evidence comes from experiments in which a longer exposure time was used. Exposure for 2 hr.--Following an exposure to 3H-uridine for 2 hr, cells at interphase and at all stages of mitosis were labeled (Table V). Cells at metaphase, anaphase, and telophase showed fewer grains than interphase and prophase cells. They also showed a greater relative number of grains over the cytoplasm than interphase or prophase cells: i.e., the cells in division showed a lower nuclear/cytoplasmic ratio of grains than interphase or prophase cells. In squash preparations, the cytoplasm occupies an area 4-6 times greater than the nucleus. Thus, in interphase cells the total number of grains is not only greater over the nuclei than over the cytoplasm, but they are concentrated into the relatively smaller area occupied by the nucleus. Interphase cells from the longest treatment show the highest grain counts (Table V, cf. Tables II and IV) and they also show the lowest nucleus/cytoplasm ratio of Experimental
Cell Research
35
322
D. Davidson
grains. This is in agreement with the evidence of nuclear synthesis of RNA and its subsequent movement to the cytoplasm. There was no evidence that metaphase cells had incorporated RNA into their chromosomes to any extent during the preceding prophase, though there must have been an abundant supply of labeled RNA, as judged by the grain counts of early prophase cells following a I-hr treatment (Table IV). Thus there is evidence from these results that RNA does not become a chromosome constituent in late prophase. A study was also made of the distribution of the grains in the cytoplasm of cells at anaphase. The grains over the cytoplasm were not localized in the midspindle region but were distributed at random, evidence that RNA was not shed from the chromosomes at anaphase. It may be noted that the change in the ratio of grains (nucleus/cytoplasm) would not be so marked if there was a rapid catabolic breakdown of cytoplasmic RNA during metaphase and anaphase; such a breakdown has been described [25] but there is no evidence for it in the results reported here.
DISCUSSION
The synthesis of RNA, as indicated by autoradiographs, appears to occur in the nucleus throughout interphase and early and midprophase cells of V. faba. It did not appear to be localized in the nucleolus. A similar result was reported from HeLa S3 [3] and Chinese hamster cells ([23, cf., however, [26] in which sections were used). Most reports agree that the site of the synthesis is the nucleus and that RNA migrates from the nucleus to the cytoplasm ([4, 16, 23, 261; cf., however, [22, 241). The cells of Vicia roots, like those of the Chinese hamster, show that the essential requirements for RNA synthesis are an intact nucleus and chromosomes that have not entered the stage of late prophase. The chromosomes in interphase and early and midprophase function in RNA synthesis but apparently cannot do so when they are condensed into the metaphase and anaphase form; synthesis appears to be prevented by spiralization. This is shown by the failure of cells undergoing mitosis to incorporate labeled precursors (Tables I, II; refs. [20, 231). In the cells of Vicia roots, the movement into the cytoplasm of acid-insoluble RNA, which is probably a fairly highly polymerized molecule, has occurred after only a 30-min treatment with tritiated precursors (Tables i, II). This movement into the cytoplasm of nuclear RNA prevents the ratio of grain counts of nucleus to cytoplasm from increasing from 30-min to l-hr treatments (Tables I, III, and II, IV) and from l- to 2-hr treatments (Tables Experimental
Cell Research
35
RNA
synthesis
in roots
of Vicia
faba
323
IV, V) in both interphase and early and midprophase cells. In cells of late prophase the movement of RNA to the cytoplasm is particularly effective and is responsible for the change in nucleus/cytoplasmic. ratios from mid- to late-prophase (Tables II, IV). Late prophase cells differ from cells at earlier stages in that they are no longer synthesizing RNA; they also differ in that the RNA that was synthesized in earlier stages is lost to the cytoplasm at a very rapid rate. The rapid movement of RNA to the cytoplasm may be correlated with the breakdown of the nuclear membrane; it is also related to the failure of chromosomes, actively contracting in late prophase, to take up RNA as a constituent, except in small amounts. R,vA und the composition of the mitotic chromosome.-Grains seen over metaphase chromosomes indicate that some isotopically labeled RNA is a constituent of the mitotic chromosome. The distribution of grains at the various stages of cell development indicates, however, that at most only a small fraction of the RNA synthesized in the nucleus during prophase or late interphase is incorporated into the chromosomes. Most of the nuclear RNA moves to the cytoplasm in late prophase. This evidence does not agree with previous suggestions that there is a progressive increase in the amount of RNA on the prophase chromosomes [S, 91. The results reported here indicate that if RNA does become a chromosomal constituent in prophase, then it must either be low molecular weight RNA or must have been synthesized at least 2 hr before late prophase (and hence, non-radioactive) and stored, ready for division. Neither type of RNA would be revealed by the present experiments. La Cour has shown, using 3H-adenosine, that metaphase chromosomes of Vicia are not labeled, i.e., they do not contain RNA, though he does find labeling of metaphase chromosomes of Trillium following prolonged exposure to 3H-adenosine [13]. The completion of the process of coiling that is first seen in prophase may depend, in part, upon RNA. Such RNA could be responsible for rendering the mitotic chromosomes sensitive to RNAase [lo]. The results reported here are based on acid-insoluble RNA; RNA of a low molecular weight might be acid-soluble, though this is unlikely, and would not have been detected. Such RNA might account for the results based on double-staining techniques. Using these methods it has been found that RNA is associated with prophase chromosomes [S, 91 and that RNA is shed by the anaphase chromosome as it moves toward the pole. The shed RNA occurs between the two groups of chromosomes [7, 81; it has also been detected there by ultraviolet spectroscopy [2]. The evidence reported here is that even the small amount of labeled RNA present on the chromosome is not released during mitosis. Experimental
Cell Research
35
324
D. Davidson
RNA synthesis and the nucleolus.-The localization over the nucleolus of grains, and therefore of labeled RNA, that Woods [26] has described in Vicia was not found in the present experiment. Grains mere distributed over the whole nucleus. This was found for nuclei in cells in interphase and prophase. No acceleration of nucleolar synthesis of RNA preceding mitosis vvas seen, in particular there was no marked synthesis in the nucleolus duringearlyprophase. It was also found that incorporation of labeled precursors almost ceased before the disappearance of the nucleolus. In cells in late prophase the nucleolus is present for some time, yet at this stage there is no incorporation (Table V) of 3H- uridine. It appears that synthesis of RNA in the nucleus stops before the nucleolus disperses, and therefore cessation of RNA synthesis cannot be due to the breakdown of the nucleolus. Though the nucleolus is present towards the end of prophase, it no longer has its original spherical shape. The cessation of RNA synthesis in the nucleolus may follow the change of shape of the nucleolus, which probably marks the beginning of the separation of its constituents. Cessation of synthesis is also associated with the beginning of the breakdown of the nuclear membrane. It has been suggested [6, 141 that nucleolar protein has a structural continuity throughout the stages of mitosis. This protein may be associated with RXA. It does not appear, hovverer, that any structural continuity of RNA protein or RNA results from attachment to the nucleolus organizer of the chromosome. The metaphase chromosomes that are labeled after exposure to 3H-cytidine or 3H-uridine showed no localization of grains near the secondary constriction, which is the region of the chromosome carrying the nucleolar organizer.
SUMMARY
Roots of V. faba were grown in solutions of 3H-cytidine and 3H-uridine, RNA precursors, for 30 min, 1 hr or 2 hr. Grain counts were made over nuclei and cytoplasm in squash preparations. Ry means of these counts, synthesis of RNA was followed in interphase and dividing cells. Results indicate that: 1. RNA is synthesized in the nuclei of cells in interphase and early and middle prophase. There appears to be no synthesis in cells in mitosis. 2. Labeled RNA moves from the nuclei to the cytoplasm. 3. In late prophase, nuclear RNA is shed into the cytoplasm with the breakdown of the nuclear membrane and nucleolus. ,C. Some grains were seen over the chromosomes. Of the RNA in the nucleus in prophase, very little of it appears to become a chromosome constituent. Experimental
Cell
Research
35
RNA
synthesis
in roots of Vicia
faba
32.5
REFERENCES 1. 2. 3. 4. 5. 6. 7.
DAVIDSON, D., Chromosoma 9, DAVIES, H. G., Exptl Cell Res. FEINENDEGEN, L., Brookhaven GOLDSTEIN, L. and PLAUT, W., GOLDSTEIN, L., MICON, J. and HARRIS, H., Nature 190, 1077 JACOBSON, W. and WIIBB, M.,
8. __
Endeauour
39 (1957). 3, 453 (1952). Symp. in Biol. 12, 172 (1959). Proc. Nat1 Acud. Sci. U.S. 40, 874 (1955). CROCKER, T. T., Biochim. Biophys. Acta 45, 82 (1960). (1961). J. Physiol.
9. KAUFMANN, B. P. and DAS, N. IO. ~ Chromosoma 7, 19 (1955). Il. KAUFMANN, B. P., MCDONALD,
17. ~18. ~ 19.
20. 21. 22.
23. 24. 25. 26. 27. 28.
Carnegie
P.,
__
Wash.
Year
Book
M.
Inst.
Wash.
R., GAY, M. N., (1947). KAUFMANS, B. P., MCDONALD, M. R. and GAY, H., LA COUR, L. F., Exptl Cell Res. 29, 112 (1963). MARTIN, P. G., N~alure 190, 1078 (1961). PERRY, R. P., Exptl Cell Res. 20, 216 (1960). PRESCOTT, D. M., Exptl Cell Res. 12, 196 (1957).
Inst.
12. 13. 14. 15. 16.
112, 2P (1951).
11, 200 (1952). Year
WILSOS,
Book K.
and
52, 238 (1952-53). WYMAS,
R.,
Cnmegie
46, 141
ibid. 19, 29 (1960). J. Biophys. Biochem. Cyfol. in Proceedings of the First
Suture
162, 814 (1948).
6, 203 (1959).
IUB/IUBS International Symposium, Stockholm, Vol. 2, Biological Structure and Function, p. 527. T. W. GOODWIX and 0. LINDBERG (cds.) Academic Press, London, 1961. PRESCOTT, D. M. and BENDER, M. A., Ercpt[ Cell Res. 26, 260 (1962). RABISOVITCH, M. P. and PLAUT, W., Ez$ Cell Res. 10,120 (i956). SWIFT, H., REBHUX, L., RASCH, E. and WOODARD, J., in Cellular Mechanisms in Differentiation and Growth, p. 45. D. RUDNICK (ed.) Princeton University Press, Princeton, N.J. 1956. TAYLOR, J. H., Ann. N.Y. Acad. Sci. 90, 409 (1960). WOODARD. J., GELBER. B. and SWIFT, H.. Exptl Cell Res. 23. 258 (1961). WOODARD; J.; RASCH, E. and SWIFT, H., J. Biophys. Biochem. Cyiol. 9,‘445 (1961). WOODS, P. S., Brookhaven Symp. in Biol. 12, 153 (1959). WOODS, P. S. and TAYLOR, J. H., Lab. Znuest. 8, 309 (1959). ZALOKAR, M., Exptl Cell Res. 19, 559 (1960).
Experimenfal
Crll
Research
35
Cell Research
35, 317-325
(1964)
RNA SYNTHESIS
317
IN ROOTS OF VlCIA
FABA
D. DAVIDSON1 Biology
Division,
Oak
Ridge
National Received
Laboratory,’ July
Oak Ridge,
Term.,
U.S.A.
25, 1963
SYKTIIESIS of RNA has now been studied in a number of plant and animal cells by using radioactive precursors and autoradiography combined with such different experimental approaches as nuclear transplantation [a] and enucleation [5, 15-171. The published evidence [15, 18, 19, 21-23, 26-281 strongly supports two general conclusions: (a) RNA is synthesized in the nucleus, and (b) RNA moves from the nucleus to the cytoplasm. In some cells the site of most active synthesis within the nucleus is the nucleolus [15, 261, cf., however [3, 231. The incorporation of labeled precursors does not occur during the stages of mitosis [20, 23] when the nucleus is no longer an intact organelle. RNA has been described as a constituent of the mitotic chromosome on the basis of results from double-staining treatments combined with exposure to specific enzymes (see discussions [l, 91). The results reported here do not support the conclusion that RNA present in the prophase nucleus becomes a constituent of the prophase and metaphase chromosome; consequently, they preclude the possibility of loss of RNA from chromosomes during anaphase movement [cf. 7, 8, 11, la]. The present study was undertaken, first, to determine how the incorporation of isotopically labeled precursors into cells of I’icia roots \\-as affected by the onset of mitosis, and secondly, to estimate the fate of nuclear RKA during prophase and to determine the contribution of RNA’ to the composition of the mitotic chromosome. Evidence is presented that RNA synthesis occurs only in the nucleus and not in the cytoplasm; alsb that synthesis of RNA in the nucleus ceases suddenly toward the end of prophase and does not occw while the chromosomes are in the condensed f&m. The chromosomes appear to be capable of synthesis only when they are in the dispersed condition of interphase or in the partially condensed stage of early prophase xvhile they are contained within an intact nuclear membrane. 1 f’resent Scotland. * Operated
address: by Union
Department Carbide
of Botany, Corporation
University for the United
of St. States
Andrew, Atomic
St. Energy
Experimental
Andrew,,
Fife,
Commission. Cell Research
35
318
D. Davidson MATERIAL
AND
METHODS
Beans (Vicia f&a) were germinated and grown in aerated distilled water until lateral roots appeared. The root system was suspended in solutions of 3H-cytidine or 3H-uridine for from 30 min to 2 hr. The activity of both labeled solutions was 2 PC/ml. Immediately after exposure roots \vere fixed in chilled acetic acid-alcohol (1 : 3) overnight. They were washed and then softened by immersion for 24 hr in a 1 per cent (w/v) solution of pectinase (pH 3.9). This treatment dissolves the middle lamella between cells allowing the cells to separate when pressed very gently, The apical meristems of roots were later removed and prepared as squashes. Cold TCA (5 per cent) was used to remove acid-soluble RNA but it does not soften the cells. Good preparations with well-flattened cells were obtained by using 45 per cent (v/v) acetic acid, which softens the cells. Control roots, also exposed to 3H-cytidine or 3H-uridine were prepared as squashes and incubated in buffered RNAase (1 mg/5 cc) at room temperature for 1 hr. After this treatment, grain counts over cells were almost the same as background counts, indicating that almost all radioactive material had been removed. Slides were frozen on dr>- ice before removal of coverslips with a razor blade. Squashes were coated with a liquid emulsion (Kodak, NTB2) and exposed for 2 weeks. After photographic processing, preparations were stained in toluidine blue, dehydrated, and mounted in I-uparal. In making grain counts, correction was made for background grains by counting grains in an adjacent area equal to the area of the cell. RESULTS
Exposure for 30 min and 1 hr.-Roots were exposed to solutions of 3Hcytidine and 3H-uridine for 30 min and 1 hr. In scoring preparations of roots grown in 3H-cytidine for 1 hr, the number of grains over nuclei of different sizes was counted. Incorporation of 3H-cytidine occurred in nuclei of all sizes. If nuclear size is taken as an indicator of degree of development within the cell cycle, it appears that RNA synthesis is continuous throughout interphase. In roots exposed to 3H-cytidine for 1 hr, mean grain counts over nuclei of different sizes (average of 50 cells) were: 13.1 + 1.5 (95 per cent confidence limits) in cells with largest nuclei, 6.7 + 1.l in cells with smallest nuclei, and 14.5 * 1.02 for cells of all sizes of nuclei. The total amount of incorporation of labeled precursor was similar in both 30-min exposures (Tables 1 and 11) but was unaccountably higher when 3H-uridine rather than 3H-cytidine was supplied to the roots for 1 hr (cf. Tables Ill and IV). Apart from this difference in the actual numbers of grains per cell, the direction of change in grain counts with time or between different stages of the mitotic cycle, were similar with both compounds. After a 30-min treatment with 3H-cytidine (Table 1) or uridine (Table II), grain counts were low over cells in metaphase, anaphase, and telophase. Experimenlnl
Cell
Research
35
RNA
synthesis
in roots of Vicia
faba
It is evident that incorporation of labeled precursors is reduced in cells undergoing mitosis. In such cells, more grains occur over the cytoplasm than over the chromosomes. In cells at early prophase and interphase, from the same roots, however, the majority of grains are over the nuclei. The relative distribution of grains is given by the ratio of grains over the nucleus (or chromosomes in dividing cells) to grains over the cytoplasm. This ratio is higher in prophase and interphase cells than in dividing cells (Tables I-IV). Later it will be shown that the presence of grains over the cytoplasm of cells in mitosis is not the result of cytoplasmic synthesis of RNA; nuclear synthesis of labeled RNA in the previous prophase or interphase, followed by movement to the cytoplasm, can account for grains over the cytoplasm at metaphase. TABLE
1. Grain counts in cells of roots of V. faba exposed to 3H-cytidine for 30 minutes.
Stage Interphase Prophase Metaphase Anaphase Telophase a Mean confidence to zero.
TABLE
Total no. grains 834 282 165 61 8
no. grains per cella 8.3i- 1.1 5.6Fl.l 3.3 Al 0.9 1.2 -
number of grains per cell is given limits are given for the measurements
II.
Mean
Ratio nucleus/ cytoplasm
Total cells
3.3/l 2.711 0.4/l 0.4/l
with 95 per cent confidence of anaphase and telophase
100 50 50 50 50 limits. because
No measure of the means tend
Grain counts in cells of roots of V. faba exposed to 3H-uridine for 30 minutes.
Stage Prophase Early Middle Late Metaphase Anaphase Telophase a See footnote
Total no. grains
162 164 39 33 17 23 ‘, Table
Mean
no. grains per cella
8.lk 2.2 8.2 It 1.7 2.0 k 0.5 3.3 i 1.9 1.7 2.3
Ratio nucleus/ cytoplasm
Total cells
3.8/l 1.4/l 0.411 0.3/l 0.5/l 0.5/l
20 20 20 10 10 10
I. Ex,perimenlal
Cell Research
35
320
D. Davidson
Active incorporation of the labeled compounds occurred in prophase c.ells (Tables I-V). I n cells treated with 3H-cytidine for either 30 min or 1 hr (Tables I and III) a fall in the ratio of nuclear to cytoplasmic grain counts from 1 hr to 30 min (i.e., from interphase through the various stages of mitosis) was found; this was the first evidence of a marked loss of nuclear RNA to the cytoplasm during prophase. Prophase cells of roots that had been grown in 3H-uridine were classified as early, middle, and late prophase on the degree of chromosome contraction. It is clear that incorporation occurs in early and middle prophase but is greater in early prophase (Tables II and IV). In both treatments it is also evident that in late prophase labeled RNA moves from the nucleus to the cytoplasm, giving a changed nucleus/ cytoplasmic ratio. Thus the ratio of grain counts is 3.8/l in early prophase and 0.4/l in late prophase in cells treated with 3H-uridine for 30 min (Table II). TABLE
III.
Grain counts in cells of roots of V. faba exposed to 3H-cytidine for 1 hour. Total no. grains
Stage Interphase Prophase Metaphase Anaphase Telophase a See footnote T.~BI,E
Mean
14.5Fl.O 14.12 1.8 7.3 i 2.0 5.4k3.0 2.3 i: 1.4
2175 705 365 270 115 ‘, Table
no. grains per cella
Ratio nucleus/ cytoplasm
Total cells
2.9/l 1.2/l 0.5/l 0.5/l 0.3/l
150 50 50 50 50
I.
IV. Grain counts in cells of roots of V. faba exposed to 3H-uridine Total no. grains
Stage
no. grains per cella
Ratio nucleus/ cytoplasm
Total cells
Interphase Prophase
4193
41.9 f 6.5
1.5/l
100
Early Middle Late Metaphase Anaphase Telophase
932 903 892 844 671 430
‘16.6 + 1.4 45.2 k 6.2 44.6 IL 5.3 33.8 -t 3.5 26.8 + 3.4 17.2ri-1.9
2.0/l 1.3/l 0.7/l 0.5/l 0.6/l 0.3/l
20 20 20 25 25 25
a See footnote Experimental
lIean
for 1 hour.
Cell Research
‘, Table 35
I.
RNA
synthesis
in roots of Vicia
faba
This change of ratio and the failure to find large numbers of grains over metaphase chromosomes indicates that (a) there is little synthesis of RNA in late prophase, and (b) most of the RNA present in the nucleus moves to the cytoplasm and very little of it becomes a constituent of the metaphase chromosomes. TARLE V. Grain counts in cells of roots of V. faba exposed to 3H-uridine
Stage Interphase Prophase Metaphase Anaphase Telophase a See footnote
Total no. grains
10,810 5342 3757 3355 2592 a, Table
Mean
no. grains per cella
108.1? 106.8 75.1 67.1 51.8
k i k k
7.5 7.7 7.8 8.1 6.S
Ratio nucleus/ cytoplasm
1.4/l 1.3/l 0.7/l 0.8/l 0.3/l
for 2 hour.
Total cells
100 50 50 50 50
I.
Doubling the duration of treatments resulted in a marked increase, almost a doubling, in the mean number of grains per interphase cell (Tables I-IV). The nucleus-to-cytoplasm ratio of the mean number of grains also changes with time. It decreases slightly as the actual mean number of grains per cell increases. This change in the pattern of the distribution of labeled RNA in interphase cells appears to be in agreement with the evidence that RNA is synthesized in the nucleus and moves to the cytoplasm. Further evidence comes from experiments in which a longer exposure time was used. Exposure for 2 hr.--Following an exposure to 3H-uridine for 2 hr, cells at interphase and at all stages of mitosis were labeled (Table V). Cells at metaphase, anaphase, and telophase showed fewer grains than interphase and prophase cells. They also showed a greater relative number of grains over the cytoplasm than interphase or prophase cells: i.e., the cells in division showed a lower nuclear/cytoplasmic ratio of grains than interphase or prophase cells. In squash preparations, the cytoplasm occupies an area 4-6 times greater than the nucleus. Thus, in interphase cells the total number of grains is not only greater over the nuclei than over the cytoplasm, but they are concentrated into the relatively smaller area occupied by the nucleus. Interphase cells from the longest treatment show the highest grain counts (Table V, cf. Tables II and IV) and they also show the lowest nucleus/cytoplasm ratio of Experimental
Cell Research
35
322
D. Davidson
grains. This is in agreement with the evidence of nuclear synthesis of RNA and its subsequent movement to the cytoplasm. There was no evidence that metaphase cells had incorporated RNA into their chromosomes to any extent during the preceding prophase, though there must have been an abundant supply of labeled RNA, as judged by the grain counts of early prophase cells following a I-hr treatment (Table IV). Thus there is evidence from these results that RNA does not become a chromosome constituent in late prophase. A study was also made of the distribution of the grains in the cytoplasm of cells at anaphase. The grains over the cytoplasm were not localized in the midspindle region but were distributed at random, evidence that RNA was not shed from the chromosomes at anaphase. It may be noted that the change in the ratio of grains (nucleus/cytoplasm) would not be so marked if there was a rapid catabolic breakdown of cytoplasmic RNA during metaphase and anaphase; such a breakdown has been described [25] but there is no evidence for it in the results reported here.
DISCUSSION
The synthesis of RNA, as indicated by autoradiographs, appears to occur in the nucleus throughout interphase and early and midprophase cells of V. faba. It did not appear to be localized in the nucleolus. A similar result was reported from HeLa S3 [3] and Chinese hamster cells ([23, cf., however, [26] in which sections were used). Most reports agree that the site of the synthesis is the nucleus and that RNA migrates from the nucleus to the cytoplasm ([4, 16, 23, 261; cf., however, [22, 241). The cells of Vicia roots, like those of the Chinese hamster, show that the essential requirements for RNA synthesis are an intact nucleus and chromosomes that have not entered the stage of late prophase. The chromosomes in interphase and early and midprophase function in RNA synthesis but apparently cannot do so when they are condensed into the metaphase and anaphase form; synthesis appears to be prevented by spiralization. This is shown by the failure of cells undergoing mitosis to incorporate labeled precursors (Tables I, II; refs. [20, 231). In the cells of Vicia roots, the movement into the cytoplasm of acid-insoluble RNA, which is probably a fairly highly polymerized molecule, has occurred after only a 30-min treatment with tritiated precursors (Tables i, II). This movement into the cytoplasm of nuclear RNA prevents the ratio of grain counts of nucleus to cytoplasm from increasing from 30-min to l-hr treatments (Tables I, III, and II, IV) and from l- to 2-hr treatments (Tables Experimental
Cell Research
35
RNA
synthesis
in roots
of Vicia
faba
323
IV, V) in both interphase and early and midprophase cells. In cells of late prophase the movement of RNA to the cytoplasm is particularly effective and is responsible for the change in nucleus/cytoplasmic. ratios from mid- to late-prophase (Tables II, IV). Late prophase cells differ from cells at earlier stages in that they are no longer synthesizing RNA; they also differ in that the RNA that was synthesized in earlier stages is lost to the cytoplasm at a very rapid rate. The rapid movement of RNA to the cytoplasm may be correlated with the breakdown of the nuclear membrane; it is also related to the failure of chromosomes, actively contracting in late prophase, to take up RNA as a constituent, except in small amounts. R,vA und the composition of the mitotic chromosome.-Grains seen over metaphase chromosomes indicate that some isotopically labeled RNA is a constituent of the mitotic chromosome. The distribution of grains at the various stages of cell development indicates, however, that at most only a small fraction of the RNA synthesized in the nucleus during prophase or late interphase is incorporated into the chromosomes. Most of the nuclear RNA moves to the cytoplasm in late prophase. This evidence does not agree with previous suggestions that there is a progressive increase in the amount of RNA on the prophase chromosomes [S, 91. The results reported here indicate that if RNA does become a chromosomal constituent in prophase, then it must either be low molecular weight RNA or must have been synthesized at least 2 hr before late prophase (and hence, non-radioactive) and stored, ready for division. Neither type of RNA would be revealed by the present experiments. La Cour has shown, using 3H-adenosine, that metaphase chromosomes of Vicia are not labeled, i.e., they do not contain RNA, though he does find labeling of metaphase chromosomes of Trillium following prolonged exposure to 3H-adenosine [13]. The completion of the process of coiling that is first seen in prophase may depend, in part, upon RNA. Such RNA could be responsible for rendering the mitotic chromosomes sensitive to RNAase [lo]. The results reported here are based on acid-insoluble RNA; RNA of a low molecular weight might be acid-soluble, though this is unlikely, and would not have been detected. Such RNA might account for the results based on double-staining techniques. Using these methods it has been found that RNA is associated with prophase chromosomes [S, 91 and that RNA is shed by the anaphase chromosome as it moves toward the pole. The shed RNA occurs between the two groups of chromosomes [7, 81; it has also been detected there by ultraviolet spectroscopy [2]. The evidence reported here is that even the small amount of labeled RNA present on the chromosome is not released during mitosis. Experimental
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35
324
D. Davidson
RNA synthesis and the nucleolus.-The localization over the nucleolus of grains, and therefore of labeled RNA, that Woods [26] has described in Vicia was not found in the present experiment. Grains mere distributed over the whole nucleus. This was found for nuclei in cells in interphase and prophase. No acceleration of nucleolar synthesis of RNA preceding mitosis vvas seen, in particular there was no marked synthesis in the nucleolus duringearlyprophase. It was also found that incorporation of labeled precursors almost ceased before the disappearance of the nucleolus. In cells in late prophase the nucleolus is present for some time, yet at this stage there is no incorporation (Table V) of 3H- uridine. It appears that synthesis of RNA in the nucleus stops before the nucleolus disperses, and therefore cessation of RNA synthesis cannot be due to the breakdown of the nucleolus. Though the nucleolus is present towards the end of prophase, it no longer has its original spherical shape. The cessation of RNA synthesis in the nucleolus may follow the change of shape of the nucleolus, which probably marks the beginning of the separation of its constituents. Cessation of synthesis is also associated with the beginning of the breakdown of the nuclear membrane. It has been suggested [6, 141 that nucleolar protein has a structural continuity throughout the stages of mitosis. This protein may be associated with RXA. It does not appear, hovverer, that any structural continuity of RNA protein or RNA results from attachment to the nucleolus organizer of the chromosome. The metaphase chromosomes that are labeled after exposure to 3H-cytidine or 3H-uridine showed no localization of grains near the secondary constriction, which is the region of the chromosome carrying the nucleolar organizer.
SUMMARY
Roots of V. faba were grown in solutions of 3H-cytidine and 3H-uridine, RNA precursors, for 30 min, 1 hr or 2 hr. Grain counts were made over nuclei and cytoplasm in squash preparations. Ry means of these counts, synthesis of RNA was followed in interphase and dividing cells. Results indicate that: 1. RNA is synthesized in the nuclei of cells in interphase and early and middle prophase. There appears to be no synthesis in cells in mitosis. 2. Labeled RNA moves from the nuclei to the cytoplasm. 3. In late prophase, nuclear RNA is shed into the cytoplasm with the breakdown of the nuclear membrane and nucleolus. ,C. Some grains were seen over the chromosomes. Of the RNA in the nucleus in prophase, very little of it appears to become a chromosome constituent. Experimental
Cell
Research
35
RNA
synthesis
in roots of Vicia
faba
32.5
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Experimenfal
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