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Far-red-induced mitotic delay and the apparent increase of X-ray induced chromatid aberrations in Tradescantia microspores
Radiation
Botany,
1965,
Vol.
5, pp. 293 to 298.
Pergamon
Press Ltd.
Printed
in Great
Britain.
FAR-RED-INDUCED MITOTIC DELAY AND THE APPARENT INCREASE OF X-RAY INDUCED CHROMATID ABERRATIONS IN 2%4DESCAJVZZ4 MICROSPORES* TE HSIU Biology
Division,
Oak
MAt
Ridge
and
National (Received
SHELDON
Laboratory, 23 October
WOLFF Oak
Ridge,
Tennessee,
U.S.A.
1964)
Abstract-Experiments have been carried out that show that far-red radiation when given in conjunction with X-rays to Tradescantia microspores induces a mitotic delay that shifts the arrival of cells at metaphase. Since S and G, are parts of the cell cycle that are not uniformly sensitive to X-rays, the shifts can lead to an apparent synergistic effect of far-red radiation This is only true, however, if cell and X-rays in the induction of chromatid aberrations. samples are taken at a fixed time after X-irradiation. When many sample-s are taken at successive times to chafacterize the changes in sensitivity, the apparent synergism is seen to be merely a reflection of the shift. The use of far-red radiation to delay mitosis and, thus, to allow a recovery from X-ray-induced pycnosis has allowed the characterization of the changes in radiation sensitivity as the cells progress through the cell cycle towards metaphase. There are two peaks of sensitivity. The one closer to metaphase is usually not seen because of the radiation-induced pycnosis. R&mm&-On a rtalist des experiences qui montrent que le rayonnement rouge lointain utilise en conjonction avec les rayons X sur les microspores de Tradescantia provoque un retard mitotique qui dtplace l’arrivee des cellules en mttaphase. Comme S et G, sont des pCriodes du cycle cellulaire non uniformtment sensibles aux rayons X, ces changements peuvent conduite Bun effet synergique apparent des rayons rouges lointains et X dans l’induction d’aberrations chromatidiques. Cependant ceci est seulement vrai lorsque les Cchantillons sont prClev6 B un temps fixC aprts I’irradiation par les rayons X. Quand plusieurs echantillons sont prelev& g des moments successifs en vne de caracttriser les changements de sensibilitt on s’aperGoit que le synergisme apparent est le simple reflet du dtplacement mitotique. L’utilisation des rayons rouges lointains en vue de retarder la mitose done de permettre un recouvrement de la pycnose induite par les rayons X a permis de caracttriser les changements de sensibilitk aux radiations au tours du cycle cellulaire jusqu’en mttaphase. 11 y a deux sommets de sensibilitC. Celui qui est le plus proche de la mttaphase n’est gtn&alement pas dttectt en raison de la pycnose induite par les radiations. Zusammenfassung-Versuche an Z%a&excantia-Pollenk&nern zeigen, dass Infrarote Strahlung, wenn sie gemeinsam mit Rijntgenstrahlen gegeben wird, den Ablauf der Mitose hemmt und dadurch den Eintritt der Zellen in die Metaphase verziigert. Da die Abschnitte S und G, des Zellteilungszyklus gegen Rbntgenstrahlen nicht in gleichem Ausmass empfindlich sind, kann diese VerzGgerung zu einem scheinbaren Synergismus von Infrarot und Rijntgenstrahlen bei der Induktion von Chromatidenaberrationen f&en. Dies ist allerdings nur dann * Research sponsored by the Corporation. t Present address: Department
U.S.
Atomic
of Biological
Energy
Commission
Sciences, 293
Western
under Illinois
contract University,
with
the
Macomb,
Union Illinois.
Carbide
296
TE HSIU
MA and SHELDON
sensitivity may be seen in those cells that were close to metaphase (at ca. 15 hr). We believe that cells in this p.eak close to metaphase ordinarily would reach metaphase earlier while still pycnotic and so not be storable. The delay in mitosis seems to have allowed enough time to elapse for recovery of pycnosis so that these cells now can be scored. When experiments of this type are repeated in Traakvcantia, the time sequence seems to vary from experiment to experiment. The qualitative nature of the effect, however, is obtained each time. For instance, in Fig. z it is seen that after X-rays alone there is a single peak of aberrations
135 TIME
WOLFF
in more aberrations than X-rays alone. The reasons for this are that the peaks that occur .from 16.5 to rg hr in Tradescantia are defined by one point when fixations are made every I ‘5 hr, and we think that in Fig. 2 the point at 16.5 hr probably did not represent cells of maximum sensitivity. In other partial experiments (and in the Vicia experiments) in which we concentrated on obtaining points around the peaks, the height of the peaks was the same after X-rays or X-rays and far-red (as was found in Fig. I). MOH and WITHROW found in Vtia faba that the far-red effect was reversible by a treatment with red radiation. Such a reversal is consistent
150
16.5
18Q
AFTER
XRAYS
(hr)
195
2to
FIG. 2. Effect of far-red radiation on the yield of X-ray-induced chromatid aberrations observed at various times after X-irradiation. A ---A---A--3 hrinlightfollowed by lOORX-rays. B . ..o. ..i-J.. . 3 hr far-red followed by 100 R X-rays. C ---O---O100 R X-rays followed by 3 hr far-red. at 16.5 hr, whereas after the combination of X-rays and far-red radiation there is a peak some 3 hr later plus another peak much earlier. The time between these two peaks is much greater than in the experiment reported in Fig. I. Another apparent difference between the experiments in Fig. I and Fig. 2 is that the peaks after X-rays and far-red (at 19.5 hr) are higher than the X-ray peak. This should not be construed to indicate that far-red plus X-rays results
with the hypothesis that the far-red effect is mediated through a phytochrome of the type described by BORTHWICK and HENDRICKS(~) for various morphogenetic effects. The phytochrome can exist in two different forms : one that absorbs light of 660 rnp (red-absorbing form) and is thus converted to a second form that absorbs light at 730 rnp. The 7So-ml*-absorbing form shifts back to the 66o-rnp-absorbing form when exposed to light at 730 rnp or when kept in the dark.
FAR-RED
X-RAY
SYNERGISM
0.9
297
A
04-
105
I I2QO
I 135 TIME
1
15Q AFTER
I
1625 l&O X RAYS (hr)
I 195
I 2FO
3. Reversal of far-red dffect by red light. 6 hr light followed by 100 R X-rays. _. . _ 0 -. . - q -. . _ 3 hr far-red, followed by 3 hr red, followed by 100 RX-rays. l -a-100 R X-rays, followed by 3 hr far-red, followed by 3 hr red. FIG.
---A
---A
---
MOH and WITHROW found that reversibility occurred only when the red light was given after the far-red. In Fig. 3 we can see the results of an experiment in which inflorescences were treated with (u) X-rays alone or with (b) far-red followed by red, either before or after X-rays. All three curves are similar, indicating that red light has eliminated the shift in the mitotic cycle induced by far-red radiation.
SWANSON and SCHWARTZ'S data t8) on stage sensitivity indicated that there were marked changes in the number of exchanges, but not of isochromatid aberrations, that were induced in different parts of the cell cycle. BREWEN,@) who worked with Vicia faba, noted a similar phenomenon in that organism. Our present data fit the same pattern. In Fig. 4 are presented the data for exchanges and isochromatid breaks observed
A &[$A I 135 TIME
FIG.
4.
Yields
of exchanges
150 AFTER
165 X RAYS
180 (hr)
195
I ?I.0
and isochromatid aberrations at various X-irradiation. A: exchanges; B: isochromatid aberrations.
times after
298
TE HSIU
MA and SHELDON
in the experiment presented in curve C, Fig. I. It may be seen that the exchange yield changes considerably throughout the cell cycle but that isochromatid deletions are more stable. BREWEN ~1 has interpreted this phenomenon in terms of Wolff and Atwood’s (see WOLFF(‘)) site concept, i.e. that as the chromosomes move through G, there is an increase in the numbers of places where two individual chromosomes come close enough to one another so that, if broken, they can form an exchange. Isochromatid aberrations that are the result of isolocus breaks in two sister chromatids are independent of site number and their frequency should not change throughout G,.
CONCLUSIONS The apparent synergistic effect of far-red radiation and X-rays in inducing chromatid abberations in Traokscantia microspores has been shown to fit the same pattern as had been observed in Vicia faba root tip studies. The synergistic effect, therefore, is not caused by a far-red-induced increase in chromosome breakage or decrease in chromosome break restitution, but is merely an observational artifact that occurs because far-red radiation induces a delay of mitosis. Thus, when far-red radiation is given in conjunction with X-rays, different cells will be at metaphase (and, thus, storable at any given time after X-rays) than is the case when X-rays alone are administered. For those aberrations that are induced in parts of the cell cycle that show differential sensitivity to X-rays, such a shift could lead one to make comparisons, at any given time, between cells that came from a more sensitive stage with those that came from a less sensitive stage. Red light has been found to reverse this effect of far-red on mitotic delay.
WOLFF
REFERENCES 1. BORTHWICK H. A. and HENDRICKS S. B. (1960) Photoperiodism in Plants. Science 132, 1223-1228. 2. BREWEN J. G. (1964) Studies on the frequencies of chromatid aberrations induced by X-rays at different times of the cell cycle of Vicia faba. Genetics 50, 101-107. 3. MOH C. C. and WITHROW R. B. (1959) Nonionizing radiant energy as an agent in altering the incidence of X-ray-induced chromatid aberrations. II Reversal of the far-red potentiating effect in Vicia by red radiant energy. Radiation Research 10, 13- 19. 4. SWANSON C. P. and HOLLAENDER A. (1946) The frequency of X-ray-induced chromatid breaks in Tradescantia as modified by near infrared radiation. Proc. Svat. Acad. Sci. U.S. 32,295-302. 5. SWANSON C. P. and JOHNSTON A. H. (1954) Radiation-induced pycnosis of chromosomes and its relation to oxygen tension. Am. Naturalist 88, 425-430. 6. SWANSON C. P. and SCHWARTZ D. (1953) Effect of X-rays on chromatid aberration in air and in nitrogen. Proc. .Nat. Acad. Sci. U.S. 39, 1241-1250. 7. WOLFF S. (1961) Some postirradiation phenomena that affect the induction of chromosome aberration. J. Cellular Comb. Physiol. Suppl. I, 58, 151-162. 8. WOLFF S. and LUIPPOLD H. E. (1960) On the apparent synergistic effect of far-red and X-rays in the production of chromatid aberrations. Prop. in Photobiol. (Proc. 3rd Internatl. Congr. Photobiol.), 457-460. 9. WOLFF S. and LUIPPOLD H. E. (1965) Mitotic delay and the apparent synergism of far-red radiation and X-rays in the production of chromosomal aberrations. Photo&em. Photobiol. 4, 439446.
10. YOST T. H. JR. (1951) The frequency of X-ray induced chromosome aberrations in Tradescantia as modified by near-infrared radiation. Genetics 36, 176184.
Botany,
1965,
Vol.
5, pp. 293 to 298.
Pergamon
Press Ltd.
Printed
in Great
Britain.
FAR-RED-INDUCED MITOTIC DELAY AND THE APPARENT INCREASE OF X-RAY INDUCED CHROMATID ABERRATIONS IN 2%4DESCAJVZZ4 MICROSPORES* TE HSIU Biology
Division,
Oak
MAt
Ridge
and
National (Received
SHELDON
Laboratory, 23 October
WOLFF Oak
Ridge,
Tennessee,
U.S.A.
1964)
Abstract-Experiments have been carried out that show that far-red radiation when given in conjunction with X-rays to Tradescantia microspores induces a mitotic delay that shifts the arrival of cells at metaphase. Since S and G, are parts of the cell cycle that are not uniformly sensitive to X-rays, the shifts can lead to an apparent synergistic effect of far-red radiation This is only true, however, if cell and X-rays in the induction of chromatid aberrations. samples are taken at a fixed time after X-irradiation. When many sample-s are taken at successive times to chafacterize the changes in sensitivity, the apparent synergism is seen to be merely a reflection of the shift. The use of far-red radiation to delay mitosis and, thus, to allow a recovery from X-ray-induced pycnosis has allowed the characterization of the changes in radiation sensitivity as the cells progress through the cell cycle towards metaphase. There are two peaks of sensitivity. The one closer to metaphase is usually not seen because of the radiation-induced pycnosis. R&mm&-On a rtalist des experiences qui montrent que le rayonnement rouge lointain utilise en conjonction avec les rayons X sur les microspores de Tradescantia provoque un retard mitotique qui dtplace l’arrivee des cellules en mttaphase. Comme S et G, sont des pCriodes du cycle cellulaire non uniformtment sensibles aux rayons X, ces changements peuvent conduite Bun effet synergique apparent des rayons rouges lointains et X dans l’induction d’aberrations chromatidiques. Cependant ceci est seulement vrai lorsque les Cchantillons sont prClev6 B un temps fixC aprts I’irradiation par les rayons X. Quand plusieurs echantillons sont prelev& g des moments successifs en vne de caracttriser les changements de sensibilitt on s’aperGoit que le synergisme apparent est le simple reflet du dtplacement mitotique. L’utilisation des rayons rouges lointains en vue de retarder la mitose done de permettre un recouvrement de la pycnose induite par les rayons X a permis de caracttriser les changements de sensibilitk aux radiations au tours du cycle cellulaire jusqu’en mttaphase. 11 y a deux sommets de sensibilitC. Celui qui est le plus proche de la mttaphase n’est gtn&alement pas dttectt en raison de la pycnose induite par les radiations. Zusammenfassung-Versuche an Z%a&excantia-Pollenk&nern zeigen, dass Infrarote Strahlung, wenn sie gemeinsam mit Rijntgenstrahlen gegeben wird, den Ablauf der Mitose hemmt und dadurch den Eintritt der Zellen in die Metaphase verziigert. Da die Abschnitte S und G, des Zellteilungszyklus gegen Rbntgenstrahlen nicht in gleichem Ausmass empfindlich sind, kann diese VerzGgerung zu einem scheinbaren Synergismus von Infrarot und Rijntgenstrahlen bei der Induktion von Chromatidenaberrationen f&en. Dies ist allerdings nur dann * Research sponsored by the Corporation. t Present address: Department
U.S.
Atomic
of Biological
Energy
Commission
Sciences, 293
Western
under Illinois
contract University,
with
the
Macomb,
Union Illinois.
Carbide
296
TE HSIU
MA and SHELDON
sensitivity may be seen in those cells that were close to metaphase (at ca. 15 hr). We believe that cells in this p.eak close to metaphase ordinarily would reach metaphase earlier while still pycnotic and so not be storable. The delay in mitosis seems to have allowed enough time to elapse for recovery of pycnosis so that these cells now can be scored. When experiments of this type are repeated in Traakvcantia, the time sequence seems to vary from experiment to experiment. The qualitative nature of the effect, however, is obtained each time. For instance, in Fig. z it is seen that after X-rays alone there is a single peak of aberrations
135 TIME
WOLFF
in more aberrations than X-rays alone. The reasons for this are that the peaks that occur .from 16.5 to rg hr in Tradescantia are defined by one point when fixations are made every I ‘5 hr, and we think that in Fig. 2 the point at 16.5 hr probably did not represent cells of maximum sensitivity. In other partial experiments (and in the Vicia experiments) in which we concentrated on obtaining points around the peaks, the height of the peaks was the same after X-rays or X-rays and far-red (as was found in Fig. I). MOH and WITHROW found in Vtia faba that the far-red effect was reversible by a treatment with red radiation. Such a reversal is consistent
150
16.5
18Q
AFTER
XRAYS
(hr)
195
2to
FIG. 2. Effect of far-red radiation on the yield of X-ray-induced chromatid aberrations observed at various times after X-irradiation. A ---A---A--3 hrinlightfollowed by lOORX-rays. B . ..o. ..i-J.. . 3 hr far-red followed by 100 R X-rays. C ---O---O100 R X-rays followed by 3 hr far-red. at 16.5 hr, whereas after the combination of X-rays and far-red radiation there is a peak some 3 hr later plus another peak much earlier. The time between these two peaks is much greater than in the experiment reported in Fig. I. Another apparent difference between the experiments in Fig. I and Fig. 2 is that the peaks after X-rays and far-red (at 19.5 hr) are higher than the X-ray peak. This should not be construed to indicate that far-red plus X-rays results
with the hypothesis that the far-red effect is mediated through a phytochrome of the type described by BORTHWICK and HENDRICKS(~) for various morphogenetic effects. The phytochrome can exist in two different forms : one that absorbs light of 660 rnp (red-absorbing form) and is thus converted to a second form that absorbs light at 730 rnp. The 7So-ml*-absorbing form shifts back to the 66o-rnp-absorbing form when exposed to light at 730 rnp or when kept in the dark.
FAR-RED
X-RAY
SYNERGISM
0.9
297
A
04-
105
I I2QO
I 135 TIME
1
15Q AFTER
I
1625 l&O X RAYS (hr)
I 195
I 2FO
3. Reversal of far-red dffect by red light. 6 hr light followed by 100 R X-rays. _. . _ 0 -. . - q -. . _ 3 hr far-red, followed by 3 hr red, followed by 100 RX-rays. l -a-100 R X-rays, followed by 3 hr far-red, followed by 3 hr red. FIG.
---A
---A
---
MOH and WITHROW found that reversibility occurred only when the red light was given after the far-red. In Fig. 3 we can see the results of an experiment in which inflorescences were treated with (u) X-rays alone or with (b) far-red followed by red, either before or after X-rays. All three curves are similar, indicating that red light has eliminated the shift in the mitotic cycle induced by far-red radiation.
SWANSON and SCHWARTZ'S data t8) on stage sensitivity indicated that there were marked changes in the number of exchanges, but not of isochromatid aberrations, that were induced in different parts of the cell cycle. BREWEN,@) who worked with Vicia faba, noted a similar phenomenon in that organism. Our present data fit the same pattern. In Fig. 4 are presented the data for exchanges and isochromatid breaks observed
A &[$A I 135 TIME
FIG.
4.
Yields
of exchanges
150 AFTER
165 X RAYS
180 (hr)
195
I ?I.0
and isochromatid aberrations at various X-irradiation. A: exchanges; B: isochromatid aberrations.
times after
298
TE HSIU
MA and SHELDON
in the experiment presented in curve C, Fig. I. It may be seen that the exchange yield changes considerably throughout the cell cycle but that isochromatid deletions are more stable. BREWEN ~1 has interpreted this phenomenon in terms of Wolff and Atwood’s (see WOLFF(‘)) site concept, i.e. that as the chromosomes move through G, there is an increase in the numbers of places where two individual chromosomes come close enough to one another so that, if broken, they can form an exchange. Isochromatid aberrations that are the result of isolocus breaks in two sister chromatids are independent of site number and their frequency should not change throughout G,.
CONCLUSIONS The apparent synergistic effect of far-red radiation and X-rays in inducing chromatid abberations in Traokscantia microspores has been shown to fit the same pattern as had been observed in Vicia faba root tip studies. The synergistic effect, therefore, is not caused by a far-red-induced increase in chromosome breakage or decrease in chromosome break restitution, but is merely an observational artifact that occurs because far-red radiation induces a delay of mitosis. Thus, when far-red radiation is given in conjunction with X-rays, different cells will be at metaphase (and, thus, storable at any given time after X-rays) than is the case when X-rays alone are administered. For those aberrations that are induced in parts of the cell cycle that show differential sensitivity to X-rays, such a shift could lead one to make comparisons, at any given time, between cells that came from a more sensitive stage with those that came from a less sensitive stage. Red light has been found to reverse this effect of far-red on mitotic delay.
WOLFF
REFERENCES 1. BORTHWICK H. A. and HENDRICKS S. B. (1960) Photoperiodism in Plants. Science 132, 1223-1228. 2. BREWEN J. G. (1964) Studies on the frequencies of chromatid aberrations induced by X-rays at different times of the cell cycle of Vicia faba. Genetics 50, 101-107. 3. MOH C. C. and WITHROW R. B. (1959) Nonionizing radiant energy as an agent in altering the incidence of X-ray-induced chromatid aberrations. II Reversal of the far-red potentiating effect in Vicia by red radiant energy. Radiation Research 10, 13- 19. 4. SWANSON C. P. and HOLLAENDER A. (1946) The frequency of X-ray-induced chromatid breaks in Tradescantia as modified by near infrared radiation. Proc. Svat. Acad. Sci. U.S. 32,295-302. 5. SWANSON C. P. and JOHNSTON A. H. (1954) Radiation-induced pycnosis of chromosomes and its relation to oxygen tension. Am. Naturalist 88, 425-430. 6. SWANSON C. P. and SCHWARTZ D. (1953) Effect of X-rays on chromatid aberration in air and in nitrogen. Proc. .Nat. Acad. Sci. U.S. 39, 1241-1250. 7. WOLFF S. (1961) Some postirradiation phenomena that affect the induction of chromosome aberration. J. Cellular Comb. Physiol. Suppl. I, 58, 151-162. 8. WOLFF S. and LUIPPOLD H. E. (1960) On the apparent synergistic effect of far-red and X-rays in the production of chromatid aberrations. Prop. in Photobiol. (Proc. 3rd Internatl. Congr. Photobiol.), 457-460. 9. WOLFF S. and LUIPPOLD H. E. (1965) Mitotic delay and the apparent synergism of far-red radiation and X-rays in the production of chromosomal aberrations. Photo&em. Photobiol. 4, 439446.
10. YOST T. H. JR. (1951) The frequency of X-ray induced chromosome aberrations in Tradescantia as modified by near-infrared radiation. Genetics 36, 176184.