- Email: [email protected]
The incorporation of 3H-thymidine by developing root tip cells
Experimental
THE
Cell Research
20, 361-367
INCORPORATION
W. Depcwtment
A.
JENSEN,
(1960)
361
OF 3H-THYMIDINE ROOT
TIP
L. G.
KAVALJIAN
BY DEVELOPING
CELLS’ and
of Botany, University of California, Berkeley, Sacramento State College, Sacramerzto, Received
August
S. MARTINOT
and Department Calif., U.S.A.
of Life
Sciences,
2, 1959
THE importance
of deoxyribose nucleic acid (DNA) in the genetics of the cell can hardly be doubted although the actual relation of DNA to gene expression, particularly in cell differentiation, is still little understood. One approach to this problem is the measurement of DKA content and synthesis in rapidly developing cells, as in the root tip. Determinations of amount of DXB in consecutive 100 p sections of onion root tips can be made and the data expressed per cell. Such data, while quantitative, give a static and generalized picture of DNA distribution, i.e., the amount of DNA per cell may be the same in two sections but for difl’erent reasons which are not detectable by this method. ‘The use of tritium (3H) labeled thymidine combined with autoradiographs permits the study of DNA synthesis in the various tissues in different stages of development. When the results from these two procedures are compared an interesting picture of the relation of DNA to cell development can be demonstrated. MATERIALS
AND
METHODS
The sets of Alfium cepa var. White Globe were placed on vials of distilled water and placed in the dark at 25°C. After 24 hours the roots had grown to about 5-10 mm in length. The sets were then transferred to vials containing 3H-thymidine 50 pG/ml for either 24 or 48 hours. They were then frozen, dehydrated by freeze-substitution, paraffin infiltrated, and sectioned at 5 ,u. Autoradiographs were made using the liquid emulsion procedure [4] and the sections stained with Azure B. The number of labeled and unlabeled nuclei in each tissue at 14 selected levels of the first 2 mm of the root were counted and the results expressed as per cent. The morphology and DNA content of this region of the onion root tip has been investigated [5,6] but it was found that, probably as a result of changes in the growing conditions, the rootcap was shorter, elongation was more pronounced, and the distribution of cell divisions had changed slightly. These differences necessitated a new the
’ This L’.S.
work was supported in part Public Health Service.
by a grant
(C-3656)
from
the National
Experimental
Cancer
Cell
Institute
of
Research
20
362
I’V. A. Jensen, L. G. Kavaljian
morphological and mitotic analysis as dures for each of these are the same as DNA measurements the sections from for each determination thus increasing
and S. Martinet
well as new DNA measurements. The procedescribed previously 15, 61 except that for the twelve roots, rather than six, were combined the accuracy of the analysis.
RESULTS
The number of nuclei showing incorporation of 3H-thymidine, cspressed as the per cent of nuclei present, is shown in Fig. 1. Also presented in Fig. 1 is the amount of DNA per cell, the per cent of cell division and the mean cell length per section. The per cent of nuclei labeled and the per cent of cell division for the root cap, protoderm, cortex, and provascular tissues arc given in Fig. 2. The data presented in Fig. 1 and 2 are for the 48 hour period in 3H-thymidine; the same pattern was obtained for the 24 hour exposure but at a lower percentage level. The data can be summarized as follo\vs: (1) Between 300 and 800 ,U from the tip (Fig. 1) there is a positive correlation between the per cent of labeled nuclei, the per cent of mitoses, and the amount of DSB per cell. The per cent of labeled nuclei is almost exactI\ twice the per cent of cells in division. This relationship is the logical result of both daughter nuclei being labeled in the course of a division (Fig. 3 f; ~1). (2) Between 800 and 2000 ,U from the tip (Fig. 1) there is an inverse correlation between per cent of labeled nuclei and the per cent mitotic frequency decreases, the per cent of labeled frequency, i.e., as the mitotic nuclei increases. The labeled nuclei in this region are apparently the result of two factors. One is the metabolic stability of 1)NA. Suclci with lalwlcd
Fig. l.-The amount of DNA per cell and per cent of nuclei labeled with 3H-thymidine of the root tip of Allium cepa as a function of the distance of the section analyzed from the tip. Also presented are the per cent of division and the average cell length per section. Experimental
Cell Research
20
Incorporation
of thymidine
by cells
363
DNA produced during mitosis retain that DSA so that nuclei labeled between 250-800 ,u are still labeled between 800-2000 ,u. The second factor is the formation of DNA by cells which will not divide again and are located SOO2000 ,L&from the tip. The increase in the percentage of labeled nuclei from the root tip to 800 ,U represents the number of nuclei labeled as a result of 70’
ROOT
CAP
PROTODERM
.
LABELED
0
MITOTIC
I3
14 I5
NVCLE, FREO.
60.
20
O\
IO 0 w n
I
2
3
4
5
6
, 6
7
, 9
YL IO
II
12
16
. . 17 19
. 1923
70-
CORTEX
PROVASCULAR
60.
50’
40’
30.
y/ . Tp;yy>k o
I
2
3
4
5
6
7
6
9
10 II
12
n , y;t, . {Q;yx\ 13 14
I5 16 17 DISTANCE
I6
1920 FROM
’ TIP
I IN
2 3 100’S
4
5 OF
6 7 MICRONS
6
9
,. . IO
I,
I2
I3
14
15
16
17 16
19 20
Fig. 2.-The per cent of nuclei labeled with 3H-thymidine and the mitotic frequency of the root cap, protoderm, cortex, and provascular tissues of the root tip of Allium cepa as a function of the distance of the section analyzed from the tip. Note that the ordinate for the root cap graph is different than the ordinates of the other graphs.
cell division, while the subsequent increase in the percentage of labeled nuclei above the level obtained at 800 ,B represents the number of nuclei labeled as a result of subsequent synthesis of DNA without mitosis. (3) The DNA per cell remains relatively constant from 800 to 1200 ,U from the tip (Fig. 1) as a result of a balance between the decrease in the number of nuclei with high DNA values that are going to divide and the increase in the number of nuclei with high DNA4 values that have ceased dirision. Above 1200 ,U the balance is lost by the almost complete cessation of 24 - 60173252
Experimental
Cell
Research
20
W. il. Jensen, L. G. Kavaljian
and S. iV!arsinot
cell division while the synthesis of DNA by the cells continues apparently undiminished. Thus, while cell division ceases, DSA synthesis does not. (4) The increase in labeled nuclei between 800 and 2000 ,U from the tip (Fig. 1) can also be roughly correlated with cell elongation \vhich begins at approximately 800 ,M from the tip while the DNA amount begins to increase at approximately 1200-1300 ,U from the tip. This correlation is probably not causal in the sense that DNA synthesis results in elongation hut that both may be controlled or influenced by the same factors. (5) While all the tissues present in this portion of the root follow the general pattern (Fig. l), each tissue has a distinctive pattern of its o\vn (Fig. 2). This is true both for the distribution of the labeled nuclei and of the divisions. (6) The apical initials, that occur 300 ,u from the tip of the root, contained no cells in division and no labeled nuclei (in Fig. 1 the percentage of labeled nuclei at 300 ,u includes the apical initials and surrounding root cap and for this reason the total lack of labeled nuclei in the apical initials is not apparent on the graph.). This is in complete agreement \vith the work of Clo\ves [2, 31 and of *Jensen and Kavaljian [61 and again illustrates the metabolic inactivity of these cells. The unlabeled nuclei of the apical initials can be clearly seen in Figs. 3 Q and 3 b. (7) The cells of the root cap shorn a peak in number of cell divisions basal to and surrounding the apical initials. The large number of labeled nuclei (Fig. 3c) in comparison to the relatively small number of divisions indicates large amounts of DNA synthesis by cells that will not subsequently undergo division. This is in agreement with the measurements of DNA per nucleus using the Feulgen procedure made by Swift [9] that indicate high C values for the root cap. (8) The epidermis shows less nuclear labeling in the basal sections (1000 to 2000 ,u from the tip) than either the cortex or the provascular tissue. This result could mean that for the majority of epidermal cells the cessation of division is accompanied by a cessation of DNA synthesis. The epidermis is the only tissue analyzed here that shows this t?pe of pattern. The synthesis of DNA does not stop completely, however, and there is steady, though slight, increase in the basal sections. (9) The cortex shows a pattern very similar to the average (Fig. 1). This is to be expected since over 80 per cent of any section is composed of cortical cells [Sl. The synthesis of DSAI in the cells of the cortex clearly continues after the cessation of cell division. A general view of the cortex is shown in the non-median longitudinal section illustrated in Fig. 3d. (10) The provascular tissue contains a relatively high number of cells. Experimenfnl
Cell Research
20
Incorporation
of thymidine
by cells
Fig. 3.--AIlium ceprc \\:hitc Globe, 10 ,u sections of the root tip stained with Azure B. A, ;IIedian I.s. of tip region showing unlabeled nuclei of apical initials, j, 150; II, X.S. of tip regiou showing unlabeled nuclei of apical initials, x 272; C, X.S. of root cap. x 109; D, nowmedian 1,s. in (he region of elongation showing general distribution of labclrti nuclei primarily in the cortex, I 68; E, 1,s. showing the elongated. labeled nuclei of vascular cells, x 272; I’, 1,s. showing a labeled mitotic figure---tclophase, y 330; G, 1,s. showing a labeled mitotic figure-anaphaw, h 330; II, 1,s. iu the cortex showing unlabeled nucleoli of labeled nuclei, :i 135.
I\-ith lahcled nuclei in the most apical sections. In this region (350-500 1~ from the tip), the percentage of cell divisions is less than half the percentage of labeled nuclei indicating that nuclei other than those just about to diCtie are synthesizing DSA. The work of Swift [C3] indicates high DNA levels for nuclei of vascular cells. The present data could mean that 1)S.I synthesis
366
W. A. Jensen, L. G. Kavaljian
and S. Martinot
resulting in high DNA per nuclei values is occurring very close to the root tip. The elongated nuclei characteristic of many vascular cells are usuall) heavily labeled, Fig. 3e. (11) Many of the nuclei in the basal sections appeared to be very heavily labeled. An attempt was made to count nuclei showing heavy incorporation but this was abandoned as unproductive because of the large number of sectioned nuclei and the problem of self-absorption of the tritium. In the heavily labeled nuclei it is clear that the nucleoli are completely lacking in active thymidine, Fig. 311. This suggests that the nucleoli are not active in DNA synthesis. DISCUSSION
The relation of DNA synthesis to cell development which develops from the data presented here and earlier [5, 61 is an interesting one and can be outlined as follows. The cells of the apical initials divide infrequently and there is little DNA synthesis. Basal to the apical initials the cells begin to increase in diameter and to divide frequently and DNA synthesis occurs in more and more cells as the number of mitoses increases. As the number of cells in division reaches a peak and the cells approach their final diameter, elongation begins but DNA synthesis appears unaffected. When the cells cease dividing and thus stop halving the product of DNA synthesis, the amount of DN4 per cell is eventually doubled as a result of this continued synthesis. The point that clearly stands out from the present data is that once DNA synthesis has @arted, this synthesis will continue apparently undiminished through the various stages of cell development and division. As DNA synthesis assumes a constancy, interest focuses on the mechanism of distribution of DNA, i.e., cell division. The beginning of cell elongation appears independent of cell division, although once elongation begins the number of divisions decreases. The number of cells in division during radial enlargement and early elongation are equal. The question is whether or not DNA synthesis and cell division are linked causally to cell elongation. \\‘hile the anslyer is still not completely clear, it would appear from this and previous work [Ci] that they are not causally related but that both are controlled or influenced by the same factors. The present data actually represent an extension of the earlier work on onion root tips [6] although in both cases the total length studied was 2 mm. Differences in both the growing conditions and the onion sets used, resulted Experimental
Cell Research
20
Incorporation
of fhymidine
by cells
367
in the coverage of a wider range of cellular development in the present Avork. The data presented earlier are actually equivalent to the first 1300 or 1600 ,L[ of the present work. The data presented here arc in general agreement with the work of Pelt -51 and Pelt and La Cour [8]. Pelt, however, has interpreted his data to indicate that the DNA in the more basal cells is subject to turnover or exchange and that there is some relationship between this exchange and the development of the cell. We feel, on the basis of the present data, that there is no reason to assume that the DNA formed is not stable. This is in agreement with a large mass of data for other organisms using a variety of techniques [l]. It must be made clear, however, that Pelt and La Cour [S] used l’icicl faba root tips and that differences in Allium cepu and Vicia frrbo root tips are known to exist (protein N per cell [5] for example). SUMMARY Using autoradiographs, the incorporation of 3H-thymidine by the nuclei of the cells of onion root tips was studied in relation to the stage of development, the mitotic activity and the DNA content of the cell. The data indicate that once DNA synthesis begins, the cell continues this synthesis irrespective of the stage of development or mitotic activity so that by 2 mm from the tip most of the cells contain twice the amount of DNA found in the early stages of development and four times the amount present in the apical initials. The authors wish to acknowledge the preparation of the illustrations.
the assistance
of Mr. Patrick
Healey for aid in
REFERENCES 1.
BRAHCET,
J., Biochemical Cytology. Academic Press, R’ew York, 1957.
2. CLOWES, F. A. L., J. Ezpll. 3. ~ New Phytologist 55, 29 4. FICQ, A., Arch. biol. 66, 509 5. JENSEX, W. A., Exptl. Cell
Botany
7, 307
(1956).
(1956). (1955).
Research
14, 575 (1958).
6. JENSEN, W. A. and KAVALJIAN, L. G., Am. J. Botany 45, 365 (1958). 7. PELC, S. R., Lab. Znuesfigafions 8, 225 (1959). 8. PELC, S. R. and LA COUR, L. F., Experientia 15, 131 (1959). 9. SWIFT, H.. Proc. Xafl. Acad. Sci. 36, 643 (1950).
Experimental
Cell
Research
20
THE
Cell Research
20, 361-367
INCORPORATION
W. Depcwtment
A.
JENSEN,
(1960)
361
OF 3H-THYMIDINE ROOT
TIP
L. G.
KAVALJIAN
BY DEVELOPING
CELLS’ and
of Botany, University of California, Berkeley, Sacramento State College, Sacramerzto, Received
August
S. MARTINOT
and Department Calif., U.S.A.
of Life
Sciences,
2, 1959
THE importance
of deoxyribose nucleic acid (DNA) in the genetics of the cell can hardly be doubted although the actual relation of DNA to gene expression, particularly in cell differentiation, is still little understood. One approach to this problem is the measurement of DKA content and synthesis in rapidly developing cells, as in the root tip. Determinations of amount of DXB in consecutive 100 p sections of onion root tips can be made and the data expressed per cell. Such data, while quantitative, give a static and generalized picture of DNA distribution, i.e., the amount of DNA per cell may be the same in two sections but for difl’erent reasons which are not detectable by this method. ‘The use of tritium (3H) labeled thymidine combined with autoradiographs permits the study of DNA synthesis in the various tissues in different stages of development. When the results from these two procedures are compared an interesting picture of the relation of DNA to cell development can be demonstrated. MATERIALS
AND
METHODS
The sets of Alfium cepa var. White Globe were placed on vials of distilled water and placed in the dark at 25°C. After 24 hours the roots had grown to about 5-10 mm in length. The sets were then transferred to vials containing 3H-thymidine 50 pG/ml for either 24 or 48 hours. They were then frozen, dehydrated by freeze-substitution, paraffin infiltrated, and sectioned at 5 ,u. Autoradiographs were made using the liquid emulsion procedure [4] and the sections stained with Azure B. The number of labeled and unlabeled nuclei in each tissue at 14 selected levels of the first 2 mm of the root were counted and the results expressed as per cent. The morphology and DNA content of this region of the onion root tip has been investigated [5,6] but it was found that, probably as a result of changes in the growing conditions, the rootcap was shorter, elongation was more pronounced, and the distribution of cell divisions had changed slightly. These differences necessitated a new the
’ This L’.S.
work was supported in part Public Health Service.
by a grant
(C-3656)
from
the National
Experimental
Cancer
Cell
Institute
of
Research
20
362
I’V. A. Jensen, L. G. Kavaljian
morphological and mitotic analysis as dures for each of these are the same as DNA measurements the sections from for each determination thus increasing
and S. Martinet
well as new DNA measurements. The procedescribed previously 15, 61 except that for the twelve roots, rather than six, were combined the accuracy of the analysis.
RESULTS
The number of nuclei showing incorporation of 3H-thymidine, cspressed as the per cent of nuclei present, is shown in Fig. 1. Also presented in Fig. 1 is the amount of DNA per cell, the per cent of cell division and the mean cell length per section. The per cent of nuclei labeled and the per cent of cell division for the root cap, protoderm, cortex, and provascular tissues arc given in Fig. 2. The data presented in Fig. 1 and 2 are for the 48 hour period in 3H-thymidine; the same pattern was obtained for the 24 hour exposure but at a lower percentage level. The data can be summarized as follo\vs: (1) Between 300 and 800 ,U from the tip (Fig. 1) there is a positive correlation between the per cent of labeled nuclei, the per cent of mitoses, and the amount of DSB per cell. The per cent of labeled nuclei is almost exactI\ twice the per cent of cells in division. This relationship is the logical result of both daughter nuclei being labeled in the course of a division (Fig. 3 f; ~1). (2) Between 800 and 2000 ,U from the tip (Fig. 1) there is an inverse correlation between per cent of labeled nuclei and the per cent mitotic frequency decreases, the per cent of labeled frequency, i.e., as the mitotic nuclei increases. The labeled nuclei in this region are apparently the result of two factors. One is the metabolic stability of 1)NA. Suclci with lalwlcd
Fig. l.-The amount of DNA per cell and per cent of nuclei labeled with 3H-thymidine of the root tip of Allium cepa as a function of the distance of the section analyzed from the tip. Also presented are the per cent of division and the average cell length per section. Experimental
Cell Research
20
Incorporation
of thymidine
by cells
363
DNA produced during mitosis retain that DSA so that nuclei labeled between 250-800 ,u are still labeled between 800-2000 ,u. The second factor is the formation of DNA by cells which will not divide again and are located SOO2000 ,L&from the tip. The increase in the percentage of labeled nuclei from the root tip to 800 ,U represents the number of nuclei labeled as a result of 70’
ROOT
CAP
PROTODERM
.
LABELED
0
MITOTIC
I3
14 I5
NVCLE, FREO.
60.
20
O\
IO 0 w n
I
2
3
4
5
6
, 6
7
, 9
YL IO
II
12
16
. . 17 19
. 1923
70-
CORTEX
PROVASCULAR
60.
50’
40’
30.
y/ . Tp;yy>k o
I
2
3
4
5
6
7
6
9
10 II
12
n , y;t, . {Q;yx\ 13 14
I5 16 17 DISTANCE
I6
1920 FROM
’ TIP
I IN
2 3 100’S
4
5 OF
6 7 MICRONS
6
9
,. . IO
I,
I2
I3
14
15
16
17 16
19 20
Fig. 2.-The per cent of nuclei labeled with 3H-thymidine and the mitotic frequency of the root cap, protoderm, cortex, and provascular tissues of the root tip of Allium cepa as a function of the distance of the section analyzed from the tip. Note that the ordinate for the root cap graph is different than the ordinates of the other graphs.
cell division, while the subsequent increase in the percentage of labeled nuclei above the level obtained at 800 ,B represents the number of nuclei labeled as a result of subsequent synthesis of DNA without mitosis. (3) The DNA per cell remains relatively constant from 800 to 1200 ,U from the tip (Fig. 1) as a result of a balance between the decrease in the number of nuclei with high DNA values that are going to divide and the increase in the number of nuclei with high DNA4 values that have ceased dirision. Above 1200 ,U the balance is lost by the almost complete cessation of 24 - 60173252
Experimental
Cell
Research
20
W. il. Jensen, L. G. Kavaljian
and S. iV!arsinot
cell division while the synthesis of DNA by the cells continues apparently undiminished. Thus, while cell division ceases, DSA synthesis does not. (4) The increase in labeled nuclei between 800 and 2000 ,U from the tip (Fig. 1) can also be roughly correlated with cell elongation \vhich begins at approximately 800 ,M from the tip while the DNA amount begins to increase at approximately 1200-1300 ,U from the tip. This correlation is probably not causal in the sense that DNA synthesis results in elongation hut that both may be controlled or influenced by the same factors. (5) While all the tissues present in this portion of the root follow the general pattern (Fig. l), each tissue has a distinctive pattern of its o\vn (Fig. 2). This is true both for the distribution of the labeled nuclei and of the divisions. (6) The apical initials, that occur 300 ,u from the tip of the root, contained no cells in division and no labeled nuclei (in Fig. 1 the percentage of labeled nuclei at 300 ,u includes the apical initials and surrounding root cap and for this reason the total lack of labeled nuclei in the apical initials is not apparent on the graph.). This is in complete agreement \vith the work of Clo\ves [2, 31 and of *Jensen and Kavaljian [61 and again illustrates the metabolic inactivity of these cells. The unlabeled nuclei of the apical initials can be clearly seen in Figs. 3 Q and 3 b. (7) The cells of the root cap shorn a peak in number of cell divisions basal to and surrounding the apical initials. The large number of labeled nuclei (Fig. 3c) in comparison to the relatively small number of divisions indicates large amounts of DNA synthesis by cells that will not subsequently undergo division. This is in agreement with the measurements of DNA per nucleus using the Feulgen procedure made by Swift [9] that indicate high C values for the root cap. (8) The epidermis shows less nuclear labeling in the basal sections (1000 to 2000 ,u from the tip) than either the cortex or the provascular tissue. This result could mean that for the majority of epidermal cells the cessation of division is accompanied by a cessation of DNA synthesis. The epidermis is the only tissue analyzed here that shows this t?pe of pattern. The synthesis of DNA does not stop completely, however, and there is steady, though slight, increase in the basal sections. (9) The cortex shows a pattern very similar to the average (Fig. 1). This is to be expected since over 80 per cent of any section is composed of cortical cells [Sl. The synthesis of DSAI in the cells of the cortex clearly continues after the cessation of cell division. A general view of the cortex is shown in the non-median longitudinal section illustrated in Fig. 3d. (10) The provascular tissue contains a relatively high number of cells. Experimenfnl
Cell Research
20
Incorporation
of thymidine
by cells
Fig. 3.--AIlium ceprc \\:hitc Globe, 10 ,u sections of the root tip stained with Azure B. A, ;IIedian I.s. of tip region showing unlabeled nuclei of apical initials, j, 150; II, X.S. of tip regiou showing unlabeled nuclei of apical initials, x 272; C, X.S. of root cap. x 109; D, nowmedian 1,s. in (he region of elongation showing general distribution of labclrti nuclei primarily in the cortex, I 68; E, 1,s. showing the elongated. labeled nuclei of vascular cells, x 272; I’, 1,s. showing a labeled mitotic figure---tclophase, y 330; G, 1,s. showing a labeled mitotic figure-anaphaw, h 330; II, 1,s. iu the cortex showing unlabeled nucleoli of labeled nuclei, :i 135.
I\-ith lahcled nuclei in the most apical sections. In this region (350-500 1~ from the tip), the percentage of cell divisions is less than half the percentage of labeled nuclei indicating that nuclei other than those just about to diCtie are synthesizing DSA. The work of Swift [C3] indicates high DNA levels for nuclei of vascular cells. The present data could mean that 1)S.I synthesis
366
W. A. Jensen, L. G. Kavaljian
and S. Martinot
resulting in high DNA per nuclei values is occurring very close to the root tip. The elongated nuclei characteristic of many vascular cells are usuall) heavily labeled, Fig. 3e. (11) Many of the nuclei in the basal sections appeared to be very heavily labeled. An attempt was made to count nuclei showing heavy incorporation but this was abandoned as unproductive because of the large number of sectioned nuclei and the problem of self-absorption of the tritium. In the heavily labeled nuclei it is clear that the nucleoli are completely lacking in active thymidine, Fig. 311. This suggests that the nucleoli are not active in DNA synthesis. DISCUSSION
The relation of DNA synthesis to cell development which develops from the data presented here and earlier [5, 61 is an interesting one and can be outlined as follows. The cells of the apical initials divide infrequently and there is little DNA synthesis. Basal to the apical initials the cells begin to increase in diameter and to divide frequently and DNA synthesis occurs in more and more cells as the number of mitoses increases. As the number of cells in division reaches a peak and the cells approach their final diameter, elongation begins but DNA synthesis appears unaffected. When the cells cease dividing and thus stop halving the product of DNA synthesis, the amount of DN4 per cell is eventually doubled as a result of this continued synthesis. The point that clearly stands out from the present data is that once DNA synthesis has @arted, this synthesis will continue apparently undiminished through the various stages of cell development and division. As DNA synthesis assumes a constancy, interest focuses on the mechanism of distribution of DNA, i.e., cell division. The beginning of cell elongation appears independent of cell division, although once elongation begins the number of divisions decreases. The number of cells in division during radial enlargement and early elongation are equal. The question is whether or not DNA synthesis and cell division are linked causally to cell elongation. \\‘hile the anslyer is still not completely clear, it would appear from this and previous work [Ci] that they are not causally related but that both are controlled or influenced by the same factors. The present data actually represent an extension of the earlier work on onion root tips [6] although in both cases the total length studied was 2 mm. Differences in both the growing conditions and the onion sets used, resulted Experimental
Cell Research
20
Incorporation
of fhymidine
by cells
367
in the coverage of a wider range of cellular development in the present Avork. The data presented earlier are actually equivalent to the first 1300 or 1600 ,L[ of the present work. The data presented here arc in general agreement with the work of Pelt -51 and Pelt and La Cour [8]. Pelt, however, has interpreted his data to indicate that the DNA in the more basal cells is subject to turnover or exchange and that there is some relationship between this exchange and the development of the cell. We feel, on the basis of the present data, that there is no reason to assume that the DNA formed is not stable. This is in agreement with a large mass of data for other organisms using a variety of techniques [l]. It must be made clear, however, that Pelt and La Cour [S] used l’icicl faba root tips and that differences in Allium cepu and Vicia frrbo root tips are known to exist (protein N per cell [5] for example). SUMMARY Using autoradiographs, the incorporation of 3H-thymidine by the nuclei of the cells of onion root tips was studied in relation to the stage of development, the mitotic activity and the DNA content of the cell. The data indicate that once DNA synthesis begins, the cell continues this synthesis irrespective of the stage of development or mitotic activity so that by 2 mm from the tip most of the cells contain twice the amount of DNA found in the early stages of development and four times the amount present in the apical initials. The authors wish to acknowledge the preparation of the illustrations.
the assistance
of Mr. Patrick
Healey for aid in
REFERENCES 1.
BRAHCET,
J., Biochemical Cytology. Academic Press, R’ew York, 1957.
2. CLOWES, F. A. L., J. Ezpll. 3. ~ New Phytologist 55, 29 4. FICQ, A., Arch. biol. 66, 509 5. JENSEX, W. A., Exptl. Cell
Botany
7, 307
(1956).
(1956). (1955).
Research
14, 575 (1958).
6. JENSEN, W. A. and KAVALJIAN, L. G., Am. J. Botany 45, 365 (1958). 7. PELC, S. R., Lab. Znuesfigafions 8, 225 (1959). 8. PELC, S. R. and LA COUR, L. F., Experientia 15, 131 (1959). 9. SWIFT, H.. Proc. Xafl. Acad. Sci. 36, 643 (1950).
Experimental
Cell
Research
20