On the “tertiary” structure of chromosomes

~lulation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands

405

ON T H E " T E R T I A R Y " S T R U C T U R E OF CHROMOSOMES*

SHELDON W O L F F

Laboratory of Radiobiology and Department of Anatomy, University of California, San Francisco,

Calif. (u.s.A.)

(Received May 2oth, 197 o)

SUMMARY

Although we know chromosomes consist of both nucleic acids and protein, we are still unsure about the actual organization of these components in chromosomes as they are seen in mitosis. Radiation studies in which chromosome aberrations are induced at various stages of the cell cycle have indicated that chromosomes react as though they were single-stranded structures (i.e., give rise to chromosome aberrations) during most of G~ and then behave double-stranded (i.e., give rise to chromatid aberrations) during S and G~. Studies of human lymphocytes have indicated that this transition from singleness to doubleness occurs in the absence of both DNA and protein synthesis. This has been interpreted to mean that chromosomes are multistranded structures that simply loosen at the time of transition. Attempts have been made to modify this "tertiary structure" of chromosomes to gain some insight into the nature of the binding involved in the lateral organization of chromosomes. If freshly drawn human lymphocytes are treated for 15-3o min with glycerol, which can change the dielectric constants within the cell, or with urea, which can disrupt hydrogen bonds, then radiation can induce chromatid breaks in cells that ordinarily respond to give only chromosome aberrations. It seems as though hydrogen bonds and ionic bonds are involved in maintaining the "tertiary structure" of chromosomes and that this structure can be modified in living cells.

INTRODUCTION

Ionizing radiation produces chromosomal aberrations that come from chromosome breakage and the subsequent reunion of the breaks to form new configurations. Different types of aberrations, however, are induced in various parts of the cell cycle. If, for example, cells are irradiated in prophase, each chromosome reacts to radiation as though it were quadripartite, giving rise to half-chromatid aberrations. This indicates that each of the two chromatids of a chromosome is a double-stranded * Work performed under the auspices of the U.S. Atomic Energy Commission. Abbreviation: DMSO, dimethylsulfoxide.

Mutation Res., Io (197 o) 4o5-414

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SHELDON WOLFF

structure. Nevertheless, after this seemingly double cbromatid passes through anaphase and is in G1 of the next cell cycle, it reacts to radiation as though it were single-stranded, i.e., the whole G1 chromosome, which is made up of but a single chromatid, is broken. When this broken chromosome subsequently replicates, the result is an abnormal chromosome in which both chromatids are similarly affected. Since the whole chromosome is involved, this class of aberrations has been termed chromosome aberrations (SaxOn). This is in contrast to chromatid aberrations that are induced when cells in S or G,, are irradiated. At these times, the chromosome reacts as though it were double and the individual chromatids are the unit of breakage and rejoining. Studies with Vicia faba (WOLFF la, EVANS AND SAVAGE s, WOLFF AND LUIPPOLDl6),Chinese hamster ceils (Hsu et al2, MONESI et a/.l°), and human lymphocytes (WOLFF 1~) have all shown that this transition from singleness to doubleness occurs in the latter part of G~ prior to DNA synthesis. Furthermore, experiments in which human lymphocytes were treated with DNA synthesis inhibitors and protein synthesis inhibitors (WOLFF1~) have shown that this transition occurred in the absence of both DNA and protein synthesis. This information, when considered along with the fact that the chromatid looks and acts double during mitosis (see \VoLFF 1~ for review), has strengthened the postulate that the G~ chromosome is a double-stranded structure in which the subunits are so closely appressed that the chromosome reacts to radiation as though it were single. It was suggested that these subunits separate from one another iust before S so that at this time the 2 strands can be broken individually to produce chromatid aberrations (WOLFF~a). If the G~ chromosome is indeed a multistranded structure, it seemed possible to treat the living cell with agents that could disrupt the bonds involved in maintaining the close apposition of the strands. It is this level of organization of chromosome structure that is here called "tertiary structure". It seemed reasonable to see if such treatments could change the response of the G1 chromosome to radiation. If the structure could be loosened in early G,, then chromatid aberrations would be induced at this stage wherein ordinarily only chromosome aberrations are induced. MATERIALS AND METHODS

The experiments were carried out on chromosomes in lymphocytes from freshly drawn human blood. All such lymphocytes are in G1 (or Go) of the cell cycle. If the lymphocytes are put into culture in the presence of phytohemagglutinin, they become transformed, i,e., metabolic activity increases and the cells enlarge and become blast cells. Furthermore, after some 26-3o h in culture, the ceils enter S wherein DNA is replicated. The first division of such cells ordinarily occurs some 48-54 h after the initiation of culture. Second division cells usually are not seen until after 64 h (BENI>ER A N D BREWEN~). Microcultures of cells were grown according to HUNGERFORD'Ss method. Three-tenths ml of freshly drawn whole blood was added to 5 ml of culture medium consisting of 1.6 strength Eagle's medium with ~ strength Earle's salts, 2% PHAM (Difco), ioo ~g/ml penicillin and 5 °/~g/ml streptomycin. In various experiments, the medium was supplemented with compounds that might modify the "milieu intfrieur" of the living cells so as to affect the chemical bonding that holds the Mutation Res., io (197 o) 4o5-414

ON THE TERTIARY STRUCTURE OF CHROMOSOMES

407

chromosome together. As early as 5 min after p u t t i n g the cells into the medium, t h e y were i r r a d i a t e d with 3o0 k V p X - r a y s from a G.E. Maxitron, h.v.1. 1.65 m m Cu, IOO R / m i n . A f t e r i r r a d i a t i o n , the cells were washed twice with either H a n k s ' b a l a n c e d salt solution or saline F, a n d then r e c u l t u r e d in m e d i u m free of the a d d e d compounds. A p p r o x i m a t e l y 4 h before fixation, colcemid was a d d e d to each culture to reach a final c o n c e n t r a t i o n of lO -6 M. Cells were then exposed at room t e m p e r a t u r e to a o.o75 M KCl solution for 4 min to spread chromosomes and fixed i m m e d i a t e l y in 3 : I a b s o l u t e m e t h y l alcohol:glacial acetic acid. Cytological p r e p a r a t i o n s were m a d e b y d r o p p i n g cells onto wet slides and staining with Giemsa. T h e slides were

Fig. ~. A, chromatid break; ]3, asymmetrical chromatid exchange; C, chromatid break a~zd chromosome dicentric with its associated acentric fragment; D, displaced chromatid break. Mutation Res., Io (~97o) 4o5-4~4

408

SHELDON WOLFF

scored for the aberrations induced by the radiation. The aberrations were classified as either chromosome aberrations of various types or chromatid aberrations (deletions or exchanges). Fig. I shows the types of chromatid aberrations found in these experiments. Chromatid gaps were not included in the scoring. RESULTS

All treatments utilized had to be gentle enough to ensure cell survival and allow the cells to transform and proceed through the proliferative cycle to metaphase where the chromosomes could be analyzed. The first compound used was glycerol which is thought to be capable of changing the dielectric constants within cells (H. STERN, personal communication). In the first experiment with this compound (Table I) freshly drawn blood was placed in medium containing either 5 or lO% glycerol for 5-3o min before being irradiated. Several things m a y be noticed in the data. TABLE EFFECT

I OF GLYCEROL

ON S E P A R A T I O N

OF CHROMOSOMAL

SUBUNITS

IN HUMAN

LYMPHOCYTES

Cells c o l l e c t e d b e t w e e n 4 8 - 6 5 h in c u l t u r e . N u m b e r s in p a r e n t h e s e s a r e p e r c e n t a g e s .

Preirradiation treatment Glycerol Time (rain) (,,,o)

Dose Number (R) of c e l l s

o~

5 5

5 15

5 to

3° 5

I(;

I5

to

30

Number of aberrations Chromosome Dicentvics Acentric Interstitial Terminal and rings rings deletions deletions

O

IOO

200 2o0 2o0

200 IOO IOO

96 (48 ) 17 16

O

2o0 200 200 2oo

Ioo IOO too 200

18 13 13 5 t (25.5)

O

17 (8.5) I 3 4 I 2 5 (2.5)

O

25 ( I 2 . 5 ) 8 II 8 6 II 7 (3,5)

2

_ _ _

Chromatid Deletions Exchange O

O

19 (9.5) 6 5

3 (I.5) 3 5

o o I

5 6 6 5 (2-5)

~ 7 5 13 (6.5)

I o I 1 (o.5)

First, when freshly drawn blood is irradiated, mainly chromosome aberrations are induced. The yield of chromatid aberrations found in this particular experiment was 1.5% which is not different from the spontaneous yield found in the various experiments performed in the course of this study. Furthermore, we have found that even when cells are irradiated after 21 h in culture, only chromosome aberrations are induced, though the yield of these aberrations is somewhat higher than that induced within the first few hours. Second, glycerol is an excellent radioprotective compound. It decreased the yield of chromosome dicentrics and rings by a factor of 3 to 4. Third, even though glycerol was radioprotective and decreased the yield of chromosome aberrations, a I5-min treatment with 5% glycerol led to an increase in induced chromatid breaks. The yield rose from the spontaneous level to 7%, indicating that glycerol could affect the tertiary structure of Gt chromosomes so that they now could respond to radiation as though double structures. Fourth, cells seem to be able to adjust to longer treatments with 5% glycerol. After 3 ° rain, the chromatid aberration yield was no different than that obtained without glycerol. This was confirmed in other experiments (see Table u). Fifth, treatment with IO% glycerol gave an increased yield within 5 min, and the cells were unable to adjust within ~,$lutation Res., IO (197 o) 4 o 5 - 4 1 4

409

ON T H E T E R T I A R Y S T R U C T U R E OF CHROMOSOMES TABLE

I[

EFFECT OF GLYCEROL ON SEPARATION OF CHROMOSOMAL SUBUNITS IN HUMAN LYMPHOCYTES 150 R of X - r a y s ; cells f i x e d a f t e r 54 h in c u l t u r e .

Preirradiation treatment

Number

of cells

Compound

Time (rain)

Number of aberrations Chromosome

Chromatid

Dicentrics dcentric and rings rings

lnterstilial Terminal deletions

deletions

Deletions Exchange

-25% Hanks' 25% Hanks'

-I 5

ioo 5° 5°

34 37 37

i 3 2

i2 6 4

5 4 4

2 o i

i o o

5% glycerol 5% glycerol

15 60

ioo ioo

25 21

2 3

9 5

4 8

9 2

o o

30 rain. Treatment with 5% glycerol for 15 rain or lO% glycerol for 5, 15, or 30 min all gave a statistically significant increase in the numbers of radiation-induced chromatid breaks. It should be noted that glycerol also caused red cells to haemolyse. Glycerol evidently crosses the cell membrane and lowers the osmotic pressure so that water can flow into the cell. Therefore, to make certain that the effect of glycerol was not caused simply by hypotonieity within the irradiated cell, the experiments reported in Table II were performed. Whole blood was suspended in 1/4 strength Hanks' balanced basic salts solution or in 5% glycerol in medium and then irradiated. The cells could not be kept in Hanks' solution for long periods of time because they lose viability. In any case, treatment with 1/4 strength Hanks' solution for 5 min did not affect the yield of chromatid aberrations. Treatment with 5% glycerol for 15 rain again raised the yield of chromatid deletions. The cells were able to adjust to a longer period of treatment as evidenced by the fact that at 60 min the yield of deletions was the same as that induced in the controls. Since the cells were irradiated in the presence of glycerol, the possibility TABLE

III

EFFECT OF GLYCEROL ON LYMPHOCYTES CONTROLS

SEPARATION

OF

CHROMOSOMAL

SUBUNITS

1N U N I R R A D I A T E D

HUMAN

o R of X - r a y s ; c e l l s f i x e d a f t e r 54 h in c u l t u r e .

Treatment

Number of cells Chromatid aberrations Deletions Exchanges

--*

IOO

2

5% glycerol, 5 min

IOO

I

I r r a d i a t e d (200 I~), 5°//o g l y c e r o l 5 rain 15 r a i n 30 m i n

ioo ioo ioo

2 2

I r r a d i a t e d (200 R), l O % g l y c e r o l 5 rain 15 rain 3° min

IOO IOO IOO

3 4 2

~'

800

16 =

* This control group also had one chromosome

2.0%

dicentric.

Mutation Res., i o (197o) 4 o 5 - 4 1 4

4Io

SHELDON WOLFF

existed that some component of the irradiated glycerol-medium mixture became radiomimetic and caused chromosome breakage. This was tested by putting whole blood into medium, medium with glycerol, or the medium with glycerol mixture that had been irradiated. The results presented in Table I I I show that there was no effect caused by keeping cells in the irradiated medium for differing lengths of time. 16 chromatid breaks were found in the 8oo unirradiated cells treated in these various ways giving a spontaneous aberration rate for chromatid breaks of ~.o°/'o. An attempt was made to increase the yield of chromatid exchanges by increasing the dose of radiation and thus increasing the number of breaks capable of interacting with one another to form exchanges. The number of chromosome aberrations was increased but not the number of chromatid aberrations (Table IV). This is interpreted to be an indication that, for the cell to survive, the treatment must 'FABLE EFFECT

IV OF I O ~ i I G L Y C E R O L ON S E P A R A T I O N

OF C H R O M O S O M A L 5 U B U N I T S

IN H U M A N LYMPI-IOCYTES

Cells f i x e d a f t e r 58 h in c u l t u r e .

Time of treatment

Dose ( N)

Number of cells

(~ni~)

d berrations Chromosom-e Dicentrics and rings

~hr~tnatid De~et[o~

--Ex-cha~ges

. . . . Percent -

I5

O

IOO

o

2

0

2.0

~5 3°

300

ioo

300

~oo

5° 68

5 6

~ ~

7 .0 8.0

be sufficiently gentle so that it will not cause extensive separation of the subunits. Thus, most of the genome still responds to radiation as though it were single, i.e., is thought not to loosen. Nevertheless, very short treatments with glycerol do seern to cause some of the subunits to loosen partially and thus respond to radiation as though double and give rise to chromatid aberrations. Among the types of bonds that we might expect to be affected if the dielectric constants within the cell are changed are hydrogen bonds. Consequently, an experiment was performed in which cells were treated for either 15 or 30 rain with 2 °:/O urea, which is known to disrupt hydrogen bonds. A 3o-min treatment with 2% urea led to a dramatic increase in the number of chromatid aberrations induced by 2oo rad (Table V). Subsequently, blood was treated with various concentrations of urea TA BLE EFFECT

V O F 2(~'o U R E A ON S E P A R A T I O N

OF C H R O M O S O M A L S U B U N I T S IN H U M A N L Y M P H O C Y T E S

5o cells p e r p o i n t ; f i x e d i~fter 54 h in c u l t u r e .

Treatment

Percent Chromatid aberrations Deletions Exchanges _

U r e a ~5 r a i n o R zoo R

_

o 4

o .l

o

o

16

2

U r e a 3 ° lnin o l)~ 200

R

31utation Res., IO (197 ° ) 4 o 5 - 4 1 4

_

ON T H E T E R T I A R Y

STRUCTURE

OF CHROMOSOMES

411

and either 2oo or 3oo R of X-ray. The results presented in Table VI show that ~°/o urea is not effective in separating the subunits. A 3o-min treatment with 2% urea, however, does increase the yield with a I5-min treatment leading to an intermediate value. 5 or IOyo urea, however, give a significantly higher yield of chromatid breaks with as little as a I5-min treatment. Unlike glycerol, urea did not prove to be a TABLE

VI

EFFECT OF UREA ON THE SEPARATION OF CHROMOSOMAL SUBLTNITS Cells f i x e d a f t e r 54 h in c u l t u r e ;

Treatment (~¢rea)

o%

Dose (R)

3o0

i o o cells p a r p o i n t .

Number of cells

Aberrations Chromosome Dicentrics and rings

Chromatid Deletions Exchanges

Percent

IOO

123

3

o

3.o

2o0

ioo

42

3

-

3 .o

200

IOO

22

2

--

2.O

200 200

ioo ioo

3° 51

2 I2

2 i

6.0 t4.o

min min min rain

2o0 200 3oo 3oo

ioo ioo IOO ioo

47 62 119 lO8

6 12 6 6

2 2

6.o 12.O IO.O to.o

lO% 15 r a i n

30o

ioo

156

8

I

Io.o

I °/o /

15 r a i n 3° min

~% 15 r a i n 3° inin

5% 15 3o 15 3°

--

potent radioprotective agent. Consequently, it appears that the ability of these reagents to separate the chromosomal subunits is independent of their ability to act as radioprotectors. DMSO is a relatively non-toxic compound that also can disrupt hydrogen bonds. Therefore, an attempt was made to see whether or not treatment with DMSO would lead to the production of chromatid aberrations in cells irradiated in G1. Similar experiments were performed in which cells were treated with from I to I O ~ DMSO for 15-3o min. The results are presented in Table VII. It may be seen that, at higher concentrations, DMSO is also radioprotective, but not as much as glycerol. A small but consistent increase in the number of chromatid aberrations is found after DMSO treatment but is not nearly as great after either glycerol or urea. If, however, the total data are examined, it is seen that in the presence of DMSO the percentage of chromatid breaks increases to 7.2 as opposed to the 2.o°,/o control value observed in Table III. It has previously been shown (Table III) that if medium including glycerol was irradiated, no long-lived radiomimetic agent that could induce chromatid aberrations was produced. In order to ascertain whether or not this was true for the other compounds that have been found to increase the yield of chromatid breaks when freshly drawn blood is irradiated, a similar experiment was performed in which medium containing either urea, glycerol, or DMSO was irradiated with 2o0 R. Freshly drawn whole blood was then added to this medium for 3o min after which 3~l*ttation Res.,

IO (197 o) 4 o 5 - 4 1 4

412

SHELDON WOLFF

TAI3LE VII EFFECT

OF

g)~{~O

ON SEPARATION

OF CHROMOSOMAL

SUBUNITS

IN

HUMAN

LYMPHOCYTES

20o R of X - r a y s ; cells f i x e d a f t e r 5 6 - 5 S h in culture,

Treatment

Nttmber

Aberrations

(D~ISO)

of cells

. Chromosome Di~ce~trics and rings

Percent

6o

~8

30.0

2

o

3.0

I5O 15o

54 42

36.0 28.0

15 7

o 6

lO.O 4.0

i5o IOO

34 26

22-7 26.0

4 4

I

4.0 4 .0

15o t5 o

32 27

2i. 3 18.o

8 7

,

O~o

.

. -

. . . Chromatid D~e{eh=o~s- lSxcha~iges

Per~e.~it

~

~% 15 m i n 3o r a i n O/ 5 eO

~5 m i n 3o rain iO o/ ,0

I5 rain 3 o rain

5-3 6.0

1

time the cells were washed twice and recultured. The results presented in Table VIII show that none of the irradiated media caused chromatid deletions. Since chromosomes from S cells already respond to radiation by giving ri~e to chromatid aberrations, it was necessary to ascertain whether or not treatment with these compounds could induce untransformed lymphocytes to enter S preTABLE

Vlll

LACK

EFFECT

OF

OF CHROMATID

OF

IRRADIATED

MEDIUM

INCLUDING

UREA,

GLYCEROL,

OR

D M S O oN

INDUCTION

BREAKS

Cells incubi.ted for 3 ° rain in irradiated m e d i u m (2oo R) b e f o r e r e c u l t u r i n g . Cells f i x e d a f t e r 54 h in c u l t u r e . .

.

.

.

C o m p o u n d add~'d to m e d i u m

Number of cells

Chromatid deletions

5% 5% 5~ IO°/~

i i I I

urea

ioo

glycerol

lOO

DMSO DMSO

15o IOO

maturely and partially replicate their DNA during the times that these experiments were performed. Ordinarily, unstimulated lymphocytes from non-leukemic individuals do not undergo DNA synthesis (Bo~D et al.3). Consequently, irradiated (2oo R) medium was prepared with 5% urea, 5°,/0 glycerol, or lO% glycerol. I #Ci/ml of 2 Ci/mmole tritiated thymidine was added to each 5-ml culture and o.3 ml of blood added. The cells were incubated for 3o min, washed twice with saline F and fixed. The total time for treatment, washing, and fixation was approximately 2 h. Autoradiograms were prepared with Ilford L4 liquid emulsion diluted I : I in water, iooo lymphocytes were scored for the presence or absence of label in the nuclei in each treatment. With the exception of I nucleus found in the 5% glycerol treatment, none of the 3ooo cells observed were labeled. It may be concluded that the compounds that cause chromosomes to change their response to radiation and give rise to chro.Xlulalion Res., IO (197 o) 4 o 5 - 4 1 4

ON THE TERTIARY STRUCTURE OF CHROMOSOMES

413

matid aberrations do not do this by stimulating the cells toundergopremature DNA synthesis. DISCUSSION

Although it is frequently maintained that the eukaryotic chromosome is analogous to the genophore of prokaryotes in the sense that both consist of but a single DNA double helix, there is much evidence indicating that the eukaryotic chromosome is a multistranded structure (see WOI~FE14 for review). The evidence for multistrandedness is both observational and experimental. Chromosomes look multistranded in both fixed and living (BAJEI~1) conditions. Furthermore, they respond to radiation in late G1 and during prophase as though they were doublestranded. The labeling patterns obtained in successive divisions after flash labeling the chromosomes during only one cell cycle also indicate the multistranded nature of chromosomes. Thus, L A C O U R AND P E L C 9, PEACOCK al, D E A V E N AND STUBBLE~IELD~, and DARLINGTON AND HAQUE~ have all observed isolabeling of chromatids in divisions where one would expect only one of a pair of sister chromatids to be labeled. The arguments for believing the chromosomal subunits are not simply the single polynucleotide chain of the DNA double helix, have been presented elsewhere (WoLEEI~). If the chromosome is indeed a multistranded structure, then the fact that this structure is held together (~r) loosely during metaphase and anaphase (when half chromatids are present), (2) tightly in early G~ (when the chromosome responds to radiation as though single), and (3) loosely again at the end of G1 (when the chromosome responds to radiation as though double) indicates that the tertiary structure of the chromosome must change during the cell cycle. The present experiments have shown that this tertiary structure can be partially modified by treating cells with agents that might be expected to change the dielectric constants within the cell and/or affect hydrogen bonds. We thus believe that the tertiary structure is partially maintained by hydrogen and/or ionic bonds. Therefore, treatments with glycerol, urea, or perhaps DMSO can increase the number of chromatid aberrations induced by radiation to a value that is some 5-6 times above the spontaneous yield. Some experiments have shown as much as 2 o ~ chromatid breaks (0.2 breaks per cell) after some of these treatments. It consequently appears that we can modify the way in which chromosomes respond to radiation and by a judicious choice of agents obtain information regarding the bonds involved in maintaining chromosome structure. ACKNOWLEDGEMENTS

The author wishes to thank Mrs. DOREEN SMITH and Mrs. BRITA HEWITT for their technical assistance. REFERENCES i BAJER, A,, S u b c h r o m a t i d s t r u c t u r e of c h r o m o s o m e s in t h e living state, Chromosoma, 17 (1965) 291-3o2. 2 BENDER, ~/[. A., AND J. G. BREWEN, F a c t o r s influencing chronlosonle a b e r r a t i o n yields in t h e h u m a n peripheral l e u k o c y t e s y s t e m , Mutation Res., 8 (1969) 383-399. 3 BOND, V. P., E. P. CRONKITE, T. M. FLIEDNER AND P. SCHORK, D e o x y r i b o n u c l e i c acid ~Vlutation Res., Io (197 o) 4o5-414

414

SHELDON WOLFF

s y n t h e s i z i n g cells in peripheral blood of n o r m a l h u u l a n beings, Science, i28 (1958) 202-203. 4 DARLINGTON, C. D., AND A. HAQUE, T h e replication a n d division of polynenlic c h r o m o s o m e s , Heredity, 24 (1969) 273-280. 5 DEAVEN, L. L., AND E. STUBBLEFIELD, Segregation of c h r o m o s o m a l D N A in Chinese h a m s t e r fibroblasts in vitro, Exptl. Cell Res., 55 (1969) 132-135. 6 EVANS, H. J., AND ]. R. K. SAVAGE, T h e relation b e t w e e n D N A s y n t h e s i s a m l c h r o m o s o m e s t r u c t u r e as resolved b y X - r a y d a m a g e , J. Cell Biol., 18 (1963) 525-54o. 7 l:~su, T. C., W. C. DEWEY AND 1{. M. HUMPHREY, R a d i o s e n s i t i v i t y of cells of Chinese h a m s t e r in vitro in relation to t h e cell cycle, Exptl. Cell Res., 27 (1962) 441-452. 8 bIUNGERFORD, D. 2~., L e u k o c y t e s c u l t u r e d from small inocula of whole blood a n d t h e p r e p a r a tion of m e t a p h a s e c h r o m o s o m e s b y t r e a t m e n t w i t h h y p o t o n i c KC1, Stain Technol., 4 ° (1965) 333-338. 9 LACouR, L. F., AND S. l~. PELC, Effect of colchicine on t h e utilization of labelled t h y m i d i n e d u r i n g c h r o m o s o m a l r e p r o d u c t i o n , Nature, 182 (1958) 506-508. io MONESI, V., ~I. CRIPPA AND ~1~. ZITo-BIGNAMI, T h e s t a g e of c h r o n l o s o m e d u p l i c a t i o n as revealed b y X - r a y b r e a k a g e a n d ~ H - t h y m i d i n e labeling, Chromosoma, 2i (1967) 369-386. I I PEACOCK, W. J., S u b c h r o m a t i d s t r u c t u r e a n d c h r o m o s o m e d u p l i c a t i o n in Vicia.~aba, Nature, 191 (1961) 832-833. 12 SAX, I(., A n a n a l y s i s of X - r a y i n d u c e d c h r o m o s o m a l a b e r r a t i o n s in T r a d e s c a n t i a , Genetics, 25 (194 o) 41-68. 13 WOLFF, S., T h e d o u b l e n e s s of t h e c h r o m o s o m e before D N A s y n t h e s i s as revealed b y c o m b i n e d X - r a y a n d t r i t i a t e d t h y m i d i n e t r e a t m e n t s , Radiation Res., 4 (1961) 517-518. 14 WOLFF, ~., S t r a n d e d n e s s of c h r o m o s o m e s , Intern. Rev. Cytol., 25 (19691 279-296. 15 WOLFF, S., T h e splitting of h u m a n c h r o m o s o m e s into c h r o m a t i d s iu t h e absence of eitber D N A or p r o t e i n s y n t h e s i s , Mutation Res., 8 (1969) 207 214. i6 WOLFF, S., AND H. E. L~'IVPOL~, C h r o m o s o m e s p l i t t i n g as revealed b y c o m b i n e d X - r a y a n d labeling experitnents, Exptl. Cell Res., 34 (1964) 548-356.

Mutation Res., io (i97o)~4o5 414