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The effect of centrifugation on x-ray-induced chromosomal aberrations in microspores of Tradescantia Paludosa
Radiation Botany, 1964, Vol. 4, pp. 101 t o 106. Pergamon Press Ltd. Printed in Great Britain.
THE EFFECT OF C E N T R I F U G A T I O N ON X-RAY-INDUCED CHROMOSOMAL ABERRATIONS IN MICROSPORES OF
TRADESCANTIA PALUDOSA*t EDWARD YEARGERS~ Emory University, Atlanta, Georgia (Received 1 August 1963) &bst~ct--Microspores of Tradescantia paludosa, Sax's clone No. 5, were given 400 r of X-rays at 50 r/rain at 30°C. Microscope slides were made from anther squashes after varying periods of time and cells at metaphase were scored for two-hit chromosomal aberrations. Controls centrifuged up to 10,000 × g for 8 rain, but not irradiated, showed no aberrations. When inflorescences were irradiated and centrifuged at low forces simultaneously no increase in the aberration yield (after 96 hr) was found. As centrifugal force was increased, however, the aberration yield increased non-monotonically. A second set of experiments was performed using 400 r at 50 r/rain in air. Aberrations were scored after 3, 4, and 5 days. The day in which the maximum number of aberrations was recovered shifted from the fourth to the fifth when centrifugal force exceeded 5,000 × g. The data are interpreted as showing that two processes affect aberration frequency during centrifugation. One, best described by an exponential relationship, tends to increase the aberration yield while the second, best described as linear, tends to decrease the yield of aberrations. R ~ s ~ - - - - O n a administr6 ~ des microspores de Tradescantia paludosa clone n ° 5 de Sax, une dose de 400 r ~ raison de 50 r minute ~ 30°C. Sur des pr6parations microseopiques ~ partir d'6crasement d'anth6res apr6s diff6rentes p6riodes, les aberrations chromosomiques ditopiques ont ~t~ relev~es en m~taphase. Des contr61es centrifuges jusqu'~ 10.000 g pendant 8 minutes mais non irradi6s n'ont montr6 aucune aberration. Lorsque les inflorescences sont simultandment irradides et centrifugdes faihle vitesse, on ne note aucun accroissement d'aberrations (apr6s 96 h). Cependant, lorsque la force centrifuge s'accroit, le taux d'aberrations ne s'accrolt pas d'une mani6re continue. Une seconde sdrie d'exp6riences a 6t6 r6alisde dans l'air avec 400 r ~ raison de 50 r rain. Les aberrations sont relevdes apr6s 3, 4 et 5 jours. Lejour all cours duquel le maximum d'aberrations est enregistr~ passe du 4~me au 56me jour lorsque la force centrifuge exc~de 5000 g. L'interprdtation des donn~es montre que pendant la centrifugation, deux processus affectent la frdquence des aberrations. L'un peut s'exprimer d'une mani6re optimale par une relation exponentielle; il tend ~ accroltre le taux d'aberrations alors que le second exprim6 par une relation lindaire tend ~ d~croitre le taux d'aberrations. Z u s ~ m m e ~ a s s u n g - - M i k r o s p o r e n yon Tradescantia paludosa, Sax's Klon Nr. 5, erhielten 400 r R6ntgenstrahlen (Dosisrate 50 r pro Minute, Temperatur 30°C). Nach verschiedenen Zeitabst~inden wurden Q uetschpr~iparate yon Antheren gemacht, und Zellen in Metaphase auf 2-Treffer Chromosomenaberrationen gepriift. Kontrollen, die 8 Min lang bis zu 10 000 × g zentrifugiert, aher nicht bestrabJt worden waren, zeigten keine Aberrationen. Infloreszenzen, die bestrahlt und gleichzeitig mit geringer Beschleunigung zentrifugiert worden waren, zeigten keine Steigerung der Aberrationsrate *The work described in this paper was performed as partial fulfilment of the requirements for the degree of Master of Science in Biology at Emory University in Atlanta, Georgia, under the directorship of PRoF, A. V. B~ATTV. ~Acknowledgement is made to the U.S. Atomic Energy Commission for the use of apparatus purchased under Contract No. AT (40-1) 2669. ~:Present address: Biophysics Department, Biology Research Center, Michigan State University, East Lansing, Michigan. 101
102
EFFECT OF CENTKIFUGATION ON X-RAY-INDUCED ABERRATIONS (nach 96 Stunden). Mit zunehmender Beschleunigung stieg die Aberrationsrate jedoch an, und zwar nicht monoton. In einer zweiten Versuchsserie wurden 400 r bei einer Dosisrate yon 50 r/Min in Luft gegeben. Die Aberrationen wurden nach 3, 4, und 5 Tagen ausgez~ihlt. Der Tag, an dem die maximale Zahl yon Aberrationen auftrat, verschob sich yore 4. auf den 5., wenn die Beschleunlgung 5 000 X g iiberstieg. Aus den Ergebnissen wird gesehlossen, dass zwei Prozesse die Aberrationsrate w~ihrend des Zentrifugierens beeinflussen. Der eine, den man am besten durch eine Exponentialfunktion beschreibt, l~isst die Aberrationsrate ansteigen, w~ihrend tier zweite, den man am besten durch eine lineare Funktion heschreibt, die Aberrationsrate verringert.
2080 rev/min during X-irradiation caused a higher frequency of aberrations than were found the effects of centrifugation as a mutagenic agent in uncentrifuged controls. I f the centrifugation was performed by KOSTOFF.CV) He centrifuged was delayed post-irradiation for a period greater the germinating seeds of Nicotiana, Vicia, Pisum, than five minutes no effect due to centrifugation Crepis, and wheat and found that chromosomal was found--possibly because the radiationalteration increased with rev/min and duration. induced broken ends had had time. to restitute. For the most part, treatment consisted of WOLFF and VON BORSTEL (9) using a total dose several thousand g's administered for several of 200 r were able to modify chromosome days. He found that spindle deformation aberration yields in the first microspore division occurred, chromosomes failed to separate, and in Tradescantia and in the first mitotic root-tip that chromosome dislocation occurred. division in Vicia by using centrifugation of DODSON and Yu(5) and Y u and DODSON (I°) 2300 x g before, during, and after irradiation. found that forces of less than 2,000 x g did not They found that pre-irradiation centrifugation produce detectable effects in germinating barley decreased the yield of aberrations while centriseeds. With a force of 2,000 x g for 6 hr 90 per fugation during, or after, irradiation increased cent germination was found while 200 × g for aberration yields. Cells in nitrogen before and 20 hr resulted in 75 per cent germination. A during irradiation showed no effect of postforce of 25,000 × g for 6 hr reduced germination irradiation centrifugation. This was interpreted to 30 per cent. Cytological effects included as meaning that breaks induced with anaerobic chromosomal aberrations in pollen mother cells irradiation rejoin faster than those made in air. of plants from germinated seeds. Phenotypic The purpose of the experimentation described effects included changes in awn texture, matura- by the present author is to investigate the effects tion time, and plant height. of simultaneous X-irradiation and centrifugaFRANKE and DODSON(e) found that Drosophila tion at forces between 0 x g and 10,000 x g on larvae were more resistant to centrifugation chromosomal aberrations in microspores of (85 per cent survival after 10,000 × g for 60 rain) Tradescantia paludosa. than were the adults (32 per cent survival after MATERIALS A N D M E T H O D S 10,000xg for 60 rain). Several mutants were recovered. T h e y included modification of the Inflorescences of 7-. paludosa (Sax's clone No. sex ratio, sex linked reccssives (miniature, 5) having buds with anthers containing cells in diluted eye color), autosomal recessives (bent the first microspore division were used in this wing, dwarf), and an autosomal dominant investigation. The plants were grown in a green(curly). These mutants were postulated to be house at 30°C with lighting adjusted to provide position effects based upon breakage and re- blooms during the entire year. arrangement of sections of chromosomes. T h e X-radiation was produced by a General The use of centrifugation, in conjunction Electric OX-250 industrial unit with a S. R-T. with the chromosomal aberration-producing -2 medical type tube, operating at 13 M A and effects of ionizing radiation, was first reported 250 kV and having an inherent filtration of by SAX.CS) He found that centrifugation at 3 m m of A1. Prior to each experiment the INTRODUCTION THE original English-language work describing
EDWARD YEAKGEKS X-ray machine was calibrated with a Victoreen r-meter using a 100 r thimble. A centrifuge was constructed from an International Hemacrit motor and placed in an airtight Lucite container. This container was constructed of ] in. Lucite with vents to facilitate the exchanging of gases and to allow water of a desired temperature to be pumped to a radiator inside. An opening was made in the top of the container to provide for the removal or addition of equipment or flowers. The top covering of ½ in. Lucite could be fastened down with bolts to provide an airtight closure. An aluminum centrifuge head was constructed which would accommodate about twenty inflorescences at a time lying on their sides. This arrangement prevented the centrifugal force from shearing the buds from the peduncle. Calibration of the motor was achieved by arranging a power source, a load resistor, and an oscilloscope into a series circuit. A break was made in the circuit and the two resulting wire ends were placed about 1 inch apart at the base of the aluminum centrifuge head. The head thus acted as a functional part of the circuit even while revolving. For 15 ° of each complete revolution a piece of insulating tape broke the circuit between the two "brushes" at the centrifuge head. In this manner a kind of square wave was generated at the oscilloscope. From the sweep time thus obtained, the centrifuge motor speed could be computed. The actual experimentation has been divided into two parts. All irradiation in both parts was administered at 50 r/minute for eight minutes at 30-4-2°C in air at normal atmospheric pressure. Part One: The centrifuge motor was started 5 see before the irradiation was started and the current to the centrifuge was turned off 5 see prior to shutting off the X-ray machine. The centrifuge's spinning motion was stopped by hand as soon as the box could be opened thereafter (less than 15 see after irradiation was stopped). Centrifugation was carried out at 0, 330, 520, 820, 1000, 1500, 2000, 2300, 3000, 3500, 4200, 4600, 5000, 6000, 7000, 8200, and 10,000 x g in various experiments. The inflorescences were then moved to the
103
greenhouse to a constant-temperature box at 3 0 ± 2 ° C for 96 hr prior to making slides. Part Two: All irradiation in this part was carried out under environmental conditions previously specified except that only four centrifugal forces were used: 0, 2300, 5000, and 10,000xg. Two experiments at each centrifugal force level were performed--one to be removed from the constant-temperature box for slide making after 72 hr and one after 120 hr. Data from Part I (96 hr) were also used. Temporary acetocarmine preparations were made in all cases and the two hit aberrations of the ring and dicentric t y p e s (3) w e r e scored by observing the metaphase configurations of the cells in the first microspore division. The percentage of aberrations per cell was computed and the standard error of the mean was computed from a formula given by DAHLBERO.(4) OBSERVATIONS AND RESULTS
The presentation of the experimental results obtained in this investigation is divided according to the two parts given above. Part One: Inflorescences given 400 r of Xradiation at 50 r/min in air were simultaneously centrifuged at forces ranging from 0 x g to 10,000 xg. Chromosomal aberrations were scored from metaphase configurations of the first microspore division 96 hr after irradiation. Controls at each force with no irradiation were also run. No aberrations were found in controls from 0 to 10,000xg where 100 cells were counted at each force. The data are given in Fig. 1. From 0 to 820 X g the yield was constant and a straight horizontal line was fitted to the data. From 820 to 2 3 0 0 x g and from 5000 to 10,000xg an exponential relationship was suggested by plotting the data on semi-logarithmic graph paper. Between 2300 and 5000 x g the yield fell off in a roughly parabolic fashion and a line was fitted in this region on a "pointto-point" basis. Least square techniques after the methods of ALEXAND~-R(x) were applied to the region from 8 0 0 X g to 2 3 0 0 × g and from 5 0 0 0 × g to 10,000 xg. From 800 x g to 2300 × g A----54.8 exp 0.00016 g (1) and from 5000 x g to 10,000 Xg
104
EFFECT OF CENTRIFUGATION ON X-RAY-INDUG/SD ABERRATIONS I
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•- CENTRIFUGAL FORCE(xo) Fro. 1.
A graph of per cent aberrations
(A) vs"
centrifugal force in multiples of gravity (g). The equation of region B is A = 54.6 exp 0.00016g and the equation of region C is A = 11-0 exp 0.00020 g. One hundred control cells were scored at each g force; no aberrations were found. The standard error of the mean is given. A = 11.0 exp 0.00020 g (2) where A is per cent aberrations and g is centrifugal force in g's. A comparison of the two exponential parts of equations (1) and (2) by a modified t-method for small samples (of BAILEY(2)) was made. The probability of such a difference being due to chance is 0-10 and the difference, therefore, is not considered to be significant. Part Two: This set of experiments was run to investigate the possibility that the decrease in chromosome aberrations between 2300 x g and 5000 × g was due to a delay or slowing down of the mitotic process. Plants given 400 r at 50 r/rain in air and centrifuged at either 0, 2300, 5000, or 10,000 × g simultaneously were taken from the greenhouse at 3, 4 and 5 days post-irradiation and slides were made for scoring. This yielded the time profile for chromosomal aberrations in cells going through mitosis shown in Table 1. Two hundred cells were counted on the third and fifth days while the fourth day's data is obtained from Part One. To investigate the factors responsible for the
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FIC. 2. A graph showing the difference between dotted line B (an extrapolation of A = 54-6 exp 0.00016g past 2300 × g) and the actual data in solid line C. The difference is dashed line D and rep resents a measure of the decreasein aberration yield from the maximum possible. DISCUSSION
The present investigation was initiated to extend the work of SAX(s) and WOLFF and YON BORSTEL(9) over a reasonably wide spectrum of forces. Their interpretations for the increase in X-ray-induced aberration frequency due to centrifugation was that the centrifugal forces tended to pull the broken ends apart, thus increasing the chance of illegitimate reunions. Since there is always the possibility of inducing aberrations through the effects of the centrifugation alone control experiments were performed at each force in the present investigation. The fact that the present work involved only eight minute periods of centrifugafion is believed to be the reason that no centrifugation-induced aberrations appeared after forces as high as 10,000 × g were applied. The work of KOSTOFF,(7)
EDWARD YEARGERS
105
Table I. Analysis of the time profile of the aberrant cells going through mitosis Force
in g's 0 2300 5000 10000
No. Cells % Aberrations No. Cells % Aberrations No. Cells % Aberrations Counted at at 3 days Counted at at 4 days Counted at at 5 days 3 Days 4 Days 5 Days 200 200 200 200
35.0 -4-3.3 47.0 4-3.5 26.0 4-3.0 54.5 4- 3.5
DODSON and Yu,O) Yu and DODSON(10) a n d FRANICE and DODSON(6) involved m u c h longer periods of time. Although between 0 x g and 8 0 0 X g there was no significant increase in aberrations in this investigation the aberration yield rose from 62.5 per cent at 8 2 0 x g to 80.2 per cent at 2300 X g. Evidently some obstacle was overcome around 800 x g. Perhaps some physical bond had to be broken, the chromosome ends resulting from the same break had to be separated by a m i n i m u m distance, or the chromosome ends resulting from different breaks were forced closer to one another. After dipping to a low of 29.5 per cent aberrations at 5000 x g a gradual rise is seen to 10,000xg (Fig. 1). An exponential curve was fitted to this part of the data in the same manner as for the 800 X g to 2300 X g section. Statistical analysis of the exponential terms in these two sections' of Fig. 1 revealed that the slopes were not significantly different. This suggested that a similar process might be contributing to the overall effect in both instances - - a process for which three possibilities were suggested above. The parabolic region from 2300 x g to 5000 x g could be explained as a force region in which this yield-increasing phenomenon was impaired by an unknown second phenomenon, only to be overtaken again at 5000 X g by the former effect. Alternatively, the parabolic section of the graph m a y result from a change in the number of recoverable aberrations due to a change in the rate of cell division. Part 2 of this investigation was undertaken to find out if the act of centrifugation might be decreasing the number of recoverable aberratior~ at 96 hr post-irradiation through an
800 400 400 400
62.5 4-2.5 80.2 4-2.0 29.5 4-2.3 78.5 4-2.1
200 200 200 200
49.5 4-3.5 58.5 4-3.5 49.5 4-3.5 80.1 4-2.8
effect on the rate of the nuclear cycle. T h e data of T a b l e 1 show that after 5000 x g or 10,000 × g the aberration frequency was highest in cells reaching metaphase on the fifth day after irradiation. This is directly opposed to the fact that aberration yields for cells which were subjected to 0 x g and 2300 x g were found to be largest on the fourth day after irradiation. This is interpreted as meaning that the process of centrifugation either causes radiation-damaged Tradescantia microspores in stage two or threeCa) to revert to an earlier stage and then subsequently to proceed through mitosis, or causes a temporary halting of the mitotic process, or causes a general slowing down of the mitotic process. Figure 2 shows that the difference between an extrapolation of A = 5 4 - 8 exp 0.00016g to I0,000 × g and the actual data curve over that region approximates a straight line. This is interpreted as indicating that one or more centrifugation-caused phenomena, including regression or halting of mitosis, tend to decrease the yield of recoverable X-ray-induced chromosomal aberrations in Tradescantia microspores. Furthermore, the net effect of these yielddecreasing phenomena appears to be a linear function of centrifugal force. This is the only result of this investigation which is dependent upon the assumption that the two exponential regions of the data represent the same process. Statistically, it has been shown that this is a reasonable assumption. REFERENCES
1. ALZX.A~mZR, W. E. (1961) Elements of Mathernatical Statistics. John Wiley, New York. 2. BAn~Y, N. T. J. (1959) Statistical Methods in Biology. English Universities Press, London.
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EFFECT OF C E N T K I F U G A T I O N ON X-RAY-INDUCED ABERRATIONS
3. BEATTY,J. W. and BEATTV,A. V. (1953) Duration of the stages in microspore development and in the first microspore division of Tradescantia paludosa. Am. 07. Botany 40, 593-596. 4. DAHLBERO, GUNNAR (1940) Statistical Methods for Medical and Biological Students, p. 139. Interscience Publishers, New York. 5. DOBSON,E. O. and Yu, C. K. (1958) Centrifugal force as a mutagenic agent, Preliminary report, Proc. Tenth Intern. Congr. Genet. 2, 333. 6. FR~NKE, W. E. and DOBSON, E. O. (1961) Production of mutations in Drosophila by means of centrifugal force. Can. 07. Genet. Cytol. 3, 295-299.
7. KOSTOFF, D. (1938) The effect of centrifuging upon the germinated seed from various plants. Cytologia 8, 420-442. 8. SAX, K. (1943) The effect of centrifuging upon the production of X-ray-induced chromosomal aberrations. Proc. Natl. Acad. Sci., U.S. 29, 18-21. 9. WOLFF,S. and YON BORSTEL, 1~. C. (1954) The effects of pre- and post-irradiation centrifugation on the chromosomes of Tradescantia and Vida. Proc. Natl. Acad. Sd., U.S. 40, 1138-1141. 10. Yu, C. K. and DODSON, E. O. (1960) Effects of centrifugal force upon chromosomal mutation in barley. Genetics 46, 1411-1423.
THE EFFECT OF C E N T R I F U G A T I O N ON X-RAY-INDUCED CHROMOSOMAL ABERRATIONS IN MICROSPORES OF
TRADESCANTIA PALUDOSA*t EDWARD YEARGERS~ Emory University, Atlanta, Georgia (Received 1 August 1963) &bst~ct--Microspores of Tradescantia paludosa, Sax's clone No. 5, were given 400 r of X-rays at 50 r/rain at 30°C. Microscope slides were made from anther squashes after varying periods of time and cells at metaphase were scored for two-hit chromosomal aberrations. Controls centrifuged up to 10,000 × g for 8 rain, but not irradiated, showed no aberrations. When inflorescences were irradiated and centrifuged at low forces simultaneously no increase in the aberration yield (after 96 hr) was found. As centrifugal force was increased, however, the aberration yield increased non-monotonically. A second set of experiments was performed using 400 r at 50 r/rain in air. Aberrations were scored after 3, 4, and 5 days. The day in which the maximum number of aberrations was recovered shifted from the fourth to the fifth when centrifugal force exceeded 5,000 × g. The data are interpreted as showing that two processes affect aberration frequency during centrifugation. One, best described by an exponential relationship, tends to increase the aberration yield while the second, best described as linear, tends to decrease the yield of aberrations. R ~ s ~ - - - - O n a administr6 ~ des microspores de Tradescantia paludosa clone n ° 5 de Sax, une dose de 400 r ~ raison de 50 r minute ~ 30°C. Sur des pr6parations microseopiques ~ partir d'6crasement d'anth6res apr6s diff6rentes p6riodes, les aberrations chromosomiques ditopiques ont ~t~ relev~es en m~taphase. Des contr61es centrifuges jusqu'~ 10.000 g pendant 8 minutes mais non irradi6s n'ont montr6 aucune aberration. Lorsque les inflorescences sont simultandment irradides et centrifugdes faihle vitesse, on ne note aucun accroissement d'aberrations (apr6s 96 h). Cependant, lorsque la force centrifuge s'accroit, le taux d'aberrations ne s'accrolt pas d'une mani6re continue. Une seconde sdrie d'exp6riences a 6t6 r6alisde dans l'air avec 400 r ~ raison de 50 r rain. Les aberrations sont relevdes apr6s 3, 4 et 5 jours. Lejour all cours duquel le maximum d'aberrations est enregistr~ passe du 4~me au 56me jour lorsque la force centrifuge exc~de 5000 g. L'interprdtation des donn~es montre que pendant la centrifugation, deux processus affectent la frdquence des aberrations. L'un peut s'exprimer d'une mani6re optimale par une relation exponentielle; il tend ~ accroltre le taux d'aberrations alors que le second exprim6 par une relation lindaire tend ~ d~croitre le taux d'aberrations. Z u s ~ m m e ~ a s s u n g - - M i k r o s p o r e n yon Tradescantia paludosa, Sax's Klon Nr. 5, erhielten 400 r R6ntgenstrahlen (Dosisrate 50 r pro Minute, Temperatur 30°C). Nach verschiedenen Zeitabst~inden wurden Q uetschpr~iparate yon Antheren gemacht, und Zellen in Metaphase auf 2-Treffer Chromosomenaberrationen gepriift. Kontrollen, die 8 Min lang bis zu 10 000 × g zentrifugiert, aher nicht bestrabJt worden waren, zeigten keine Aberrationen. Infloreszenzen, die bestrahlt und gleichzeitig mit geringer Beschleunigung zentrifugiert worden waren, zeigten keine Steigerung der Aberrationsrate *The work described in this paper was performed as partial fulfilment of the requirements for the degree of Master of Science in Biology at Emory University in Atlanta, Georgia, under the directorship of PRoF, A. V. B~ATTV. ~Acknowledgement is made to the U.S. Atomic Energy Commission for the use of apparatus purchased under Contract No. AT (40-1) 2669. ~:Present address: Biophysics Department, Biology Research Center, Michigan State University, East Lansing, Michigan. 101
102
EFFECT OF CENTKIFUGATION ON X-RAY-INDUCED ABERRATIONS (nach 96 Stunden). Mit zunehmender Beschleunigung stieg die Aberrationsrate jedoch an, und zwar nicht monoton. In einer zweiten Versuchsserie wurden 400 r bei einer Dosisrate yon 50 r/Min in Luft gegeben. Die Aberrationen wurden nach 3, 4, und 5 Tagen ausgez~ihlt. Der Tag, an dem die maximale Zahl yon Aberrationen auftrat, verschob sich yore 4. auf den 5., wenn die Beschleunlgung 5 000 X g iiberstieg. Aus den Ergebnissen wird gesehlossen, dass zwei Prozesse die Aberrationsrate w~ihrend des Zentrifugierens beeinflussen. Der eine, den man am besten durch eine Exponentialfunktion beschreibt, l~isst die Aberrationsrate ansteigen, w~ihrend tier zweite, den man am besten durch eine lineare Funktion heschreibt, die Aberrationsrate verringert.
2080 rev/min during X-irradiation caused a higher frequency of aberrations than were found the effects of centrifugation as a mutagenic agent in uncentrifuged controls. I f the centrifugation was performed by KOSTOFF.CV) He centrifuged was delayed post-irradiation for a period greater the germinating seeds of Nicotiana, Vicia, Pisum, than five minutes no effect due to centrifugation Crepis, and wheat and found that chromosomal was found--possibly because the radiationalteration increased with rev/min and duration. induced broken ends had had time. to restitute. For the most part, treatment consisted of WOLFF and VON BORSTEL (9) using a total dose several thousand g's administered for several of 200 r were able to modify chromosome days. He found that spindle deformation aberration yields in the first microspore division occurred, chromosomes failed to separate, and in Tradescantia and in the first mitotic root-tip that chromosome dislocation occurred. division in Vicia by using centrifugation of DODSON and Yu(5) and Y u and DODSON (I°) 2300 x g before, during, and after irradiation. found that forces of less than 2,000 x g did not They found that pre-irradiation centrifugation produce detectable effects in germinating barley decreased the yield of aberrations while centriseeds. With a force of 2,000 x g for 6 hr 90 per fugation during, or after, irradiation increased cent germination was found while 200 × g for aberration yields. Cells in nitrogen before and 20 hr resulted in 75 per cent germination. A during irradiation showed no effect of postforce of 25,000 × g for 6 hr reduced germination irradiation centrifugation. This was interpreted to 30 per cent. Cytological effects included as meaning that breaks induced with anaerobic chromosomal aberrations in pollen mother cells irradiation rejoin faster than those made in air. of plants from germinated seeds. Phenotypic The purpose of the experimentation described effects included changes in awn texture, matura- by the present author is to investigate the effects tion time, and plant height. of simultaneous X-irradiation and centrifugaFRANKE and DODSON(e) found that Drosophila tion at forces between 0 x g and 10,000 x g on larvae were more resistant to centrifugation chromosomal aberrations in microspores of (85 per cent survival after 10,000 × g for 60 rain) Tradescantia paludosa. than were the adults (32 per cent survival after MATERIALS A N D M E T H O D S 10,000xg for 60 rain). Several mutants were recovered. T h e y included modification of the Inflorescences of 7-. paludosa (Sax's clone No. sex ratio, sex linked reccssives (miniature, 5) having buds with anthers containing cells in diluted eye color), autosomal recessives (bent the first microspore division were used in this wing, dwarf), and an autosomal dominant investigation. The plants were grown in a green(curly). These mutants were postulated to be house at 30°C with lighting adjusted to provide position effects based upon breakage and re- blooms during the entire year. arrangement of sections of chromosomes. T h e X-radiation was produced by a General The use of centrifugation, in conjunction Electric OX-250 industrial unit with a S. R-T. with the chromosomal aberration-producing -2 medical type tube, operating at 13 M A and effects of ionizing radiation, was first reported 250 kV and having an inherent filtration of by SAX.CS) He found that centrifugation at 3 m m of A1. Prior to each experiment the INTRODUCTION THE original English-language work describing
EDWARD YEAKGEKS X-ray machine was calibrated with a Victoreen r-meter using a 100 r thimble. A centrifuge was constructed from an International Hemacrit motor and placed in an airtight Lucite container. This container was constructed of ] in. Lucite with vents to facilitate the exchanging of gases and to allow water of a desired temperature to be pumped to a radiator inside. An opening was made in the top of the container to provide for the removal or addition of equipment or flowers. The top covering of ½ in. Lucite could be fastened down with bolts to provide an airtight closure. An aluminum centrifuge head was constructed which would accommodate about twenty inflorescences at a time lying on their sides. This arrangement prevented the centrifugal force from shearing the buds from the peduncle. Calibration of the motor was achieved by arranging a power source, a load resistor, and an oscilloscope into a series circuit. A break was made in the circuit and the two resulting wire ends were placed about 1 inch apart at the base of the aluminum centrifuge head. The head thus acted as a functional part of the circuit even while revolving. For 15 ° of each complete revolution a piece of insulating tape broke the circuit between the two "brushes" at the centrifuge head. In this manner a kind of square wave was generated at the oscilloscope. From the sweep time thus obtained, the centrifuge motor speed could be computed. The actual experimentation has been divided into two parts. All irradiation in both parts was administered at 50 r/minute for eight minutes at 30-4-2°C in air at normal atmospheric pressure. Part One: The centrifuge motor was started 5 see before the irradiation was started and the current to the centrifuge was turned off 5 see prior to shutting off the X-ray machine. The centrifuge's spinning motion was stopped by hand as soon as the box could be opened thereafter (less than 15 see after irradiation was stopped). Centrifugation was carried out at 0, 330, 520, 820, 1000, 1500, 2000, 2300, 3000, 3500, 4200, 4600, 5000, 6000, 7000, 8200, and 10,000 x g in various experiments. The inflorescences were then moved to the
103
greenhouse to a constant-temperature box at 3 0 ± 2 ° C for 96 hr prior to making slides. Part Two: All irradiation in this part was carried out under environmental conditions previously specified except that only four centrifugal forces were used: 0, 2300, 5000, and 10,000xg. Two experiments at each centrifugal force level were performed--one to be removed from the constant-temperature box for slide making after 72 hr and one after 120 hr. Data from Part I (96 hr) were also used. Temporary acetocarmine preparations were made in all cases and the two hit aberrations of the ring and dicentric t y p e s (3) w e r e scored by observing the metaphase configurations of the cells in the first microspore division. The percentage of aberrations per cell was computed and the standard error of the mean was computed from a formula given by DAHLBERO.(4) OBSERVATIONS AND RESULTS
The presentation of the experimental results obtained in this investigation is divided according to the two parts given above. Part One: Inflorescences given 400 r of Xradiation at 50 r/min in air were simultaneously centrifuged at forces ranging from 0 x g to 10,000 xg. Chromosomal aberrations were scored from metaphase configurations of the first microspore division 96 hr after irradiation. Controls at each force with no irradiation were also run. No aberrations were found in controls from 0 to 10,000xg where 100 cells were counted at each force. The data are given in Fig. 1. From 0 to 820 X g the yield was constant and a straight horizontal line was fitted to the data. From 820 to 2 3 0 0 x g and from 5000 to 10,000xg an exponential relationship was suggested by plotting the data on semi-logarithmic graph paper. Between 2300 and 5000 x g the yield fell off in a roughly parabolic fashion and a line was fitted in this region on a "pointto-point" basis. Least square techniques after the methods of ALEXAND~-R(x) were applied to the region from 8 0 0 X g to 2 3 0 0 × g and from 5 0 0 0 × g to 10,000 xg. From 800 x g to 2300 × g A----54.8 exp 0.00016 g (1) and from 5000 x g to 10,000 Xg
104
EFFECT OF CENTRIFUGATION ON X-RAY-INDUG/SD ABERRATIONS I
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centrifugal force in multiples of gravity (g). The equation of region B is A = 54.6 exp 0.00016g and the equation of region C is A = 11-0 exp 0.00020 g. One hundred control cells were scored at each g force; no aberrations were found. The standard error of the mean is given. A = 11.0 exp 0.00020 g (2) where A is per cent aberrations and g is centrifugal force in g's. A comparison of the two exponential parts of equations (1) and (2) by a modified t-method for small samples (of BAILEY(2)) was made. The probability of such a difference being due to chance is 0-10 and the difference, therefore, is not considered to be significant. Part Two: This set of experiments was run to investigate the possibility that the decrease in chromosome aberrations between 2300 x g and 5000 × g was due to a delay or slowing down of the mitotic process. Plants given 400 r at 50 r/rain in air and centrifuged at either 0, 2300, 5000, or 10,000 × g simultaneously were taken from the greenhouse at 3, 4 and 5 days post-irradiation and slides were made for scoring. This yielded the time profile for chromosomal aberrations in cells going through mitosis shown in Table 1. Two hundred cells were counted on the third and fifth days while the fourth day's data is obtained from Part One. To investigate the factors responsible for the
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FIC. 2. A graph showing the difference between dotted line B (an extrapolation of A = 54-6 exp 0.00016g past 2300 × g) and the actual data in solid line C. The difference is dashed line D and rep resents a measure of the decreasein aberration yield from the maximum possible. DISCUSSION
The present investigation was initiated to extend the work of SAX(s) and WOLFF and YON BORSTEL(9) over a reasonably wide spectrum of forces. Their interpretations for the increase in X-ray-induced aberration frequency due to centrifugation was that the centrifugal forces tended to pull the broken ends apart, thus increasing the chance of illegitimate reunions. Since there is always the possibility of inducing aberrations through the effects of the centrifugation alone control experiments were performed at each force in the present investigation. The fact that the present work involved only eight minute periods of centrifugafion is believed to be the reason that no centrifugation-induced aberrations appeared after forces as high as 10,000 × g were applied. The work of KOSTOFF,(7)
EDWARD YEARGERS
105
Table I. Analysis of the time profile of the aberrant cells going through mitosis Force
in g's 0 2300 5000 10000
No. Cells % Aberrations No. Cells % Aberrations No. Cells % Aberrations Counted at at 3 days Counted at at 4 days Counted at at 5 days 3 Days 4 Days 5 Days 200 200 200 200
35.0 -4-3.3 47.0 4-3.5 26.0 4-3.0 54.5 4- 3.5
DODSON and Yu,O) Yu and DODSON(10) a n d FRANICE and DODSON(6) involved m u c h longer periods of time. Although between 0 x g and 8 0 0 X g there was no significant increase in aberrations in this investigation the aberration yield rose from 62.5 per cent at 8 2 0 x g to 80.2 per cent at 2300 X g. Evidently some obstacle was overcome around 800 x g. Perhaps some physical bond had to be broken, the chromosome ends resulting from the same break had to be separated by a m i n i m u m distance, or the chromosome ends resulting from different breaks were forced closer to one another. After dipping to a low of 29.5 per cent aberrations at 5000 x g a gradual rise is seen to 10,000xg (Fig. 1). An exponential curve was fitted to this part of the data in the same manner as for the 800 X g to 2300 X g section. Statistical analysis of the exponential terms in these two sections' of Fig. 1 revealed that the slopes were not significantly different. This suggested that a similar process might be contributing to the overall effect in both instances - - a process for which three possibilities were suggested above. The parabolic region from 2300 x g to 5000 x g could be explained as a force region in which this yield-increasing phenomenon was impaired by an unknown second phenomenon, only to be overtaken again at 5000 X g by the former effect. Alternatively, the parabolic section of the graph m a y result from a change in the number of recoverable aberrations due to a change in the rate of cell division. Part 2 of this investigation was undertaken to find out if the act of centrifugation might be decreasing the number of recoverable aberratior~ at 96 hr post-irradiation through an
800 400 400 400
62.5 4-2.5 80.2 4-2.0 29.5 4-2.3 78.5 4-2.1
200 200 200 200
49.5 4-3.5 58.5 4-3.5 49.5 4-3.5 80.1 4-2.8
effect on the rate of the nuclear cycle. T h e data of T a b l e 1 show that after 5000 x g or 10,000 × g the aberration frequency was highest in cells reaching metaphase on the fifth day after irradiation. This is directly opposed to the fact that aberration yields for cells which were subjected to 0 x g and 2300 x g were found to be largest on the fourth day after irradiation. This is interpreted as meaning that the process of centrifugation either causes radiation-damaged Tradescantia microspores in stage two or threeCa) to revert to an earlier stage and then subsequently to proceed through mitosis, or causes a temporary halting of the mitotic process, or causes a general slowing down of the mitotic process. Figure 2 shows that the difference between an extrapolation of A = 5 4 - 8 exp 0.00016g to I0,000 × g and the actual data curve over that region approximates a straight line. This is interpreted as indicating that one or more centrifugation-caused phenomena, including regression or halting of mitosis, tend to decrease the yield of recoverable X-ray-induced chromosomal aberrations in Tradescantia microspores. Furthermore, the net effect of these yielddecreasing phenomena appears to be a linear function of centrifugal force. This is the only result of this investigation which is dependent upon the assumption that the two exponential regions of the data represent the same process. Statistically, it has been shown that this is a reasonable assumption. REFERENCES
1. ALZX.A~mZR, W. E. (1961) Elements of Mathernatical Statistics. John Wiley, New York. 2. BAn~Y, N. T. J. (1959) Statistical Methods in Biology. English Universities Press, London.
106
EFFECT OF C E N T K I F U G A T I O N ON X-RAY-INDUCED ABERRATIONS
3. BEATTY,J. W. and BEATTV,A. V. (1953) Duration of the stages in microspore development and in the first microspore division of Tradescantia paludosa. Am. 07. Botany 40, 593-596. 4. DAHLBERO, GUNNAR (1940) Statistical Methods for Medical and Biological Students, p. 139. Interscience Publishers, New York. 5. DOBSON,E. O. and Yu, C. K. (1958) Centrifugal force as a mutagenic agent, Preliminary report, Proc. Tenth Intern. Congr. Genet. 2, 333. 6. FR~NKE, W. E. and DOBSON, E. O. (1961) Production of mutations in Drosophila by means of centrifugal force. Can. 07. Genet. Cytol. 3, 295-299.
7. KOSTOFF, D. (1938) The effect of centrifuging upon the germinated seed from various plants. Cytologia 8, 420-442. 8. SAX, K. (1943) The effect of centrifuging upon the production of X-ray-induced chromosomal aberrations. Proc. Natl. Acad. Sci., U.S. 29, 18-21. 9. WOLFF,S. and YON BORSTEL, 1~. C. (1954) The effects of pre- and post-irradiation centrifugation on the chromosomes of Tradescantia and Vida. Proc. Natl. Acad. Sd., U.S. 40, 1138-1141. 10. Yu, C. K. and DODSON, E. O. (1960) Effects of centrifugal force upon chromosomal mutation in barley. Genetics 46, 1411-1423.