428 Experimental Cell Research 41, 128-437 (1966)

S Y N C H R O N I Z A T I O N O F M A M M A L I A N C E L L S I N V I T R O B Y

I N H I B I T I O N O F T H E D N A S Y N T H E S I S

I. O P T I M A L C O N D I T I O N S 1

G. G A L A V A Z I , 2 H. S C H E N K and D. B O O T S M A 8

Medical Biological Laboratory of the National Defence Research Organization T.N.O., Rijswijk (Z.H.), The Netherlands

Received July 6, 1965

A MODERATE degree of synchrony of the life cycles of the individual cells in a population can be obtained by a single treatment with substances like 5- fluorodeoxyuridine (FUdR) [5, 6, 9, 16, 25, 29], amethopterin [14, 25, 27], thymidine (TdR) [1, 7, 33], deoxyadenosine (AdR) [33], deoxyguanosine (GdR) [19, 33], or 5-aminouracil (AU) [3, 22, 28]. A common property of these compounds is their ability to interfere with the synthesis of deoxyri- bonucleic acid (1)NA) (FUdR [10], amethopterin [12], TdR [18, 20, 23], AdR [15, 26], GdR [13, 19, 261, AU [4]). The inhibition resulting from high concentrations of exogenous T(IR, AdR, GdR or AU can be reversed by simply washing of the cells with control medium. Reversal of the effect of FUdR or amethopterin requires addition of a h)w concentration of T(tR to the cultures.

A single blocking treatment is not sufficient to obtain optimal synchrony because the cells which are in S phase at the moment of the beginning of the treatment will not be synchronized. The rationale of the double thymidine treatment, independently devised by Bootsma el al. [1] and Puck [24], is that the last treatment should preferably be started at a time when no cells are in S phase. Schematically the double treatment method can be under- stood as follows. The fraction of the population which is in S phase when the first treatment is started will not proceed further through this phase during the treatment. The other cells continue their life cycle until they are accumulated just before the S phase. Then the block of the DNA synthesis is removed and the cells are allowed to proceed through their division cycle until the last cell has finished DNA synthesis. No ceils will have reached the

1 This work was partly supported by Euratom (European Atomic Energy Community), 51-53, rue Belliard, Brussels, Belgium.

2 Present address: Research Laboratories, Department of Rheumatology, University Hospital, Leiden, The Netherlands.

3 Member of the Biology Division of Euratom.

Experimental Cell Research 41

Sgm'hronization bg inhibition of D N A sgnthesis. I 429

n e x t S p h a s e yet , if the d u r a t i o n o f S is s h o r t e r t h a n h a l f o f the to ta l c y c l e a n d

the v a r i a t i ( m in d u r a t i o n o f the cyc l e o f t he i n d i v i d u a l ce i ls is no t l a rge . At

th i s m o m e n t the s e c o n d t r e a t m e n t is s t a r t ed . N o w a l l ce l ls w i l l a c c m n u l a t e

j u s t b e f o r e the S p h a s e a n d a f t e r the e n d o f the s e c o n d t r e a t m e n t a l l ce l ls w i l l

s y n c h r o n o u s l y e n t e r this p h a s e . F r o m th is s c h e m e it is c l e a r t h a t the i n h i b i -

t ion o f the D N A s y n t h e s i s s h o u l d be as c o m p l e t e a n d as spec i f i c as p o s s i b l e ,

c o m p l e t e l y r e v e r s i b l e a n d l a s t as long as it t a k e s the s l ow e s t ce l ls to p r o c e e d

t h r o u g h the (io, M a n d (i~ p h a s e s .

I n the p r e s e n t s t u d y a n o p t i m a l p r o c e d u r e w a s e l a b o r a t e d to s y n c h r o n i z e

h u n m n ce l l s b y i n h i b i t i o n o f the D N A syn thes i s . I n c o n n e c t i o n w i t h the

d e s i r a b i l i t y to a p p l y two t r e a t m e n t s o n l y s u b s t a n c e s h a v e b e e n tes ted , the

b l o c k i n g effect o f w h i c h c a n be r e v e r s e d b y s i m p l y w a s h i n g w i t h c o n t r o l

m e d i u m .

M A T E R I A L A N D M E T H O D S

Chemicals.--Thymidine, deoxyadenos ine . H~O, deoxyguanos ine . H20 and de- o x y c y t i d i n e - H C l were purchased from California Corporat ion for Biochemical Research (grade A), 5-amino-uraci l (purum) from F luka AG and 3H-thymidine (1.9 C/m mole) from Schwarz BioResearch.

Cells. A heteroploid cell line der ived from a human k idney [30] was ma in ta ined as monolayer cul ture in silicon rubber s toppered glass bot t les (T-cell line). The growth medium consisted of 0.5 per cent l ac ta lbumin hydro lysa te and 5.5 per cent inac t iva ted newborn calf serum in Hanks ' solution, supp lemented with 100 IU penicil l in and 0.10 mg s t r ep tomyc in per ml. Vir tua l ly monocel lu lar suspensions were made by gentle t ryps in iza t ion [l] of cul tures in the logar i thmic growth per iod and seeded in 4 ml medium in modified Petr i dishes with an inner d iamete r of 45 m m [31], p rovided with a coverslip on the bo t tom. All exper iments were s ta r ted af ter 3 days incubat ion of the dishes at 37~ in an a tmosphere of humidif ied air conta in ing CO2. The cultures have then reached the logar i thmic growth phase again. The pres- sure of CO2 was control led in order to main ta in the p H of the med ium at 7.0-7.2. I t was repea ted ly ascer ta ined t ha t the cells used were not con tamina ted with PPLO.

Treatment with chemicals.--The compounds were dissolved in fresh medium jus t before use. The solutions were steri l ized by glass f i l t ra t ion, warmed to 37~ and added to the cultures af ter removal of the control medium. Solutions conta ining CdR were neut ra l ized with NaOH. Unless otherwise s ta ted , the t r ea tmen t s were t e r m ina t ed by washing the cells three t imes with control medium. All condit ions were kep t as cons tan t as possible during the exper iments . However , changes from condi t ioned to uncondi t ioned medium, cooling by a few degrees cent igrade and slight raising of the p H could not be avoided during medium rep lacement or washing. The t e rmino logy of the exper imenta l procedures is i l lus t ra ted in Fig. 1. Unless otherwise s ta ted , the Petr i dishes were inocula ted with 105 cells and the dura t ion of the t r ea t - men t s was 24 hr and of the growth periods 14 hr.

Sampling and assay of lhe populalion.--To determine the percentage of labeled cells, 0.5 t*C aH-TdlR per ml was added to the cultures. This equals a concentra t ion

Experimental Cell Research 41

430 G. Ga/avazi, H. Schenk and D. Boolsma

of 2.6 x 10-7 M TdR during the labeling period. After 20 min the cultures were washed with 0.9 per cent NaC1, fixed for at least 30 rain in a 1 : 3 acetic ac id-e thanol mixture and kept in 70 per cent ethanol unt i l the end of the experiment. Autoradio- grams were prepared with Kodak AR-10 str ipping film, exposed for 21 days, de- veloped, stained with Mayer's hematoxyl in and mounted. 1000 cells were counted

A b r,got . . . . . pt,.~ of the population

l r egutar sampling i of the population

3 d,~ys ifl control medium first first second second third

inoculation -- - -

i regu(ar sampling

I of the population

Fig. 1.--Schemes of experimental procedures. A, single treatment; B, double treatment; C~ triple treatmcnt; +, blocking of the DNA synthesis; - , dcblocking of the DNA synthesis.

in each preparation. The mid point of the labeling period was considered to be the momen t of sampling. To determine the percentage of mitoses labeled, the cultures were pulse labeled and autoradiograms were prepared at various intervals thereafter. 100-200 mitoses were counted. For determinat ions of the percentage of mitoses the cultures were fixed, stained with hema toxy l in and eosin and mounted. The n u m b e r of reliably recognizable mitoses (mid prophase to late telophase) was counted per 1000-2000 cells.

The mean count of 1 3 preparat ions was used for each kind of determinat ion. The na ture of the sampling procedure made tha t only cells adhering to the coverslips could be counted. In all preparat ions a very smal l fraction of the populat ion was pycnotic or disintegrating; these cells were not included in the counts.

Crilerion for synchrony.--The effectiveness of various synchronizing t r ea tmen t s had to be compared. The height of the peak in the curve of percentage of mitoses as plotted against t ime after t r ea tment was used as the criterion for the degree of synchrony obtained. Comparisons were always carried out in the same experiment.

Delermination of plating efficiency.--Suspensions were prepared from cultures grown in Petri dishes without coverslip and cloned using Puck 's feeder layer technique [31]. The culture medium was replaced twice during the incubat ion t ime of 13 days. The number of macroscopically visible clones was counted after fixation in Bouin ' s fluid and staining with hematoxylin. The mean count of 5-6 dishes was used for each determinat ion. The plat ing efficiency after a t r ea tmen t was expressed as per- centage of control values. Cloning capacity in control cultures of the cell line used has been found to be 98.8 per cent +29.5 (s.d.) [32].

R E S U L T S

T h e s y n c h r o n i z i n g effect of a s ingle t r e a t m e n t wi th T d R w a s f o u n d to be

h igh ly c o n c e n t r a t i o n d e p e n d e n t (Fig. 2). C o n c e n t r a t i o n s b e t w e e n 0.5 a n d

Experimenlal Cell Research 41

Sgnchroniz~tlion by itdffbilion o['DNA synthesis. I 431

15 m M were tested. The opt imal concentra t ion appeared to be 7.5 m M flw

single as well as for double treatments. Lower concentra t ions resulted in

lower peaks in lhe curve of the mitotic index. Concentrat ions higher than

7.5 mM were clearly toxic: with la mM an apl)reeiable fraction of the

- - 2 - -

, i s a 1'0 *'z 1). 16 1~

hours after end of first thymld~ne treatment

A

,~ ./

i i i i i i i

14

12 ~ : B

5 8 1 0 12 14 16 1 8

hours after end of f irst treatment

Fig. 2. Fig,. 3.

Fig . 2 . - - M i t o t i c a c t i v i t y a f t e r a s ing le t r e a t m e n t w i t h v a r i o u s c o n c e n t r a t i o n s of t h y m i d i n e . G - - O , w i t h '2 m M T d H ; � 9 1 4 9 7.5 r a M , Z I _ ~ , 15 r a M : - - , m e a n in c o n t r o l c u l t u r e s .

F ig . 3 . I M i t o t i c a c t i v i t y a f t e r a s ing le t r e a t m e n t w i t h o p t i m a l c o n c e n t r a t i o n s of s e v e r a l de- o x y r i b o n u c l e o s i d e s a n d some c o m b i n a t i o n s . A. �9 O, w i t h T d H 7.5 r a M ; � 9 1 6 9 A d H 2 m M : E I - - t l , G d R "2 mil l . /1. ~ - - - O , A d R : "l 'dR: ~ k_, G d H t T d H , - - , m e a n in c o n t r o l s .

popula t ion died and was washed away after the treatment. In the surviving

popula t ion the peak of mitotic activity came later and was lower than after a t reatment with 7.5 raM.

The same kind of concentra t ion dependence was found when the cells

were treated with other inhibitors. The opt imal concentra t ion for a single

t rea tment with AdR, (RtR or AU was 2 raM. The op t imum for AdR was

even more critical than for TdR: 2.5 m M was clearly toxic. For GdR or At ,

the optimal value was less critical than fl)r TdR. The result of a t reatment

ExperimenlaI Cell Research 41

432 G. Galavazi, H. Schenk and D. Bootsma

with CdR was also investigated. Concentrations up to 7.5 mM had no syn- chronizing effect. The synchronization obtained by a single treatment with optimal concentrations of TdR, AdR or GdR is compared in Fig. 3A. The curve of the percentage of mitoses after treatment with AU was about the same as with TdR. The origin of the differences in the time of maximal mitotic activity (Fig. 3A) was not further studied. The reproducibili ty of the effect of TdR was somewhat better and on the average the peak of mitotic activity a little higher than with AdR or GdR. The synchronizing effect of AU was about as good as of TdR, but the frequency of mitotic aberrat ions after treatment with AU was much larger than after TdR. This f requency was not quantified. On the basis of the above findings TdR was chosen for further experiments. Combinations of TdR with other inhibitors were tried, to test the possibility of obtaining improved synchronization without con- comitantly enhanced toxicity. The concentrations were for TdR, 7.5 mM and for AdR or GdR, 2 raM. The combination of TdR with AU was not tested, because treatment with AU has been found to result in interference with thymine metabolism [4]; consequently it could be expected that TdR would counteract the effect of AU. TdR +AdR and TdR + GdR had about the same effect as, respectively, AdR and GdR alone (Fig. 3A and B). TdR + AdR + GdR was extremely toxic, as was AdR + GdR. Therefore the syn- chronizing effect of a single treatment with TdR alone could not be improved

by addition of other blocking agents. The blocking effect of TdR can be reversed by washing the cells with

control medium or by addition of CdR to the inhibited culture [17, 18, 33]. The following procedures were compared to establish the optimal way of

disinhibition: (a) Addition of high concentrations of CdR to the blocked culture; (b)

washing three times followed by growth in control medium; (c) same as (b), but with an addition of 5.10-r -5 M CdR to the control medium.

Procedure a was tested with 2 and 7.5 mM CdR. Addition of CdR to a final concentration of 2 mM hardly diminished the blocking effect of 7.5 mM TdR, but 7.5 mM CdR was effective. This is also substantiated by the

finding that a treatment for 24 hr with 7.5 mM TdR + 7.5 mM CdR, followed by washing and growth in control medium, did not synchronize the popula- tion appreciably. As illustrated in Fig. 4, procedure b is clearly superior to procedure a. Addition of low concentrations of CdR to the medium used for washing and growth after the treatment (procedure c) results in the same curve of mitotic activity as with procedure b. This was chequed for single as well as for double treatments. It can thus be concluded that treatment with

Experimental Cell Research 4!

Sgnchroni:ation by inhibition of DNA synthesis. I 433

7.5 m M T d R folh,wed by three t imes wash ing with control m e d i u m is an

op t ima l bh ,ck ing and debh)eking p rocedure . In doub le t r ea tmen t expe r imen t s the op t ima l n u l n b e r of cells in the

i nocu lum was found to t,e 6.104 cells per Petri dish. Smal le r inocula resul ted

13-

12.

10-

/'\ ~o

8 -

6 -

j - - , , , , , ,

6 8 to 12 ~ 16 18

hours af ter end of f i rs t Ihymld~ne treatment

o

1 .,ko__~-_~- , ~~ / ~- ~ . . . . .

, i

hours after end of first thym,dlne treatment

l : ig. 4. Fig. 5.

I : ig. 4 . - - E f f e c t of two d i f f e r e n t w a y s of t e r m i n a t i n g a s ing le t r e a t m e n t w i t h 7.5 m M ' l 'dl~. I I - - I , m i t o t i c a c t i v i t y a f t e r w a s h i n g th ree t i m e s w i t h c o n t r o l me<i ium; ~ ~ :, a f t e r a d d i t i o n of ( :(IR to a c o n c e n t r a t i o n of 7.5 r a M ; , m e a n in con t ro l s .

F ig . 5 . - - - - l ' e rcentage of t o t a l , l a b e l e d a n d u n l a b e l e d m i t o s e s a f t e r a s ing le t r e a t m e n t w i t h 7.5 m M T d R p r e c e d e d b y l a b e l i n g w i t h a t t - ' l ' d l~ d u r i n g 21) ra in . ~ c,, t o t a l m i t o s e s ; . - , l ahele , I m i t o s e s ( c a l c u l a t e d f r o m the c o u n t e d n u m b e r of l a b e l e d m i t o s e s p e r 100 200 m i t o s e s ) ; . . , u n l a b e l e d m i t o s e s ( t h i s c u r v e is o b t a i n e d b y s u b t r a c t i o n ) .

in i r repr ( ,duc ib le growth rates, larger ones ill infer ior synchrony . The re fo re fur ther expe r imen l s were earrie(I <,tit with 6.104 instead of 105 cells as in- o c u l u m .

The op t ima l (lurati(,n ()f the inhibi t ion was ( le termined by c o m p a r i n g the

etl'ect of a single t r ea tmen t (luring 16, 20, 24, 28 or 32 hr. I )u ra t ions of 20

to 32 hr resul ted in an e(lual degree of sy lwhrony . The peak of the mitotic

index after a t r ea tmen l ()f 16 hr was mu( 'h h)wer. As op t ima l dura t ion 24 hr

was adopted .

Experimental (:ell Research 41

434 G. Gahwazi, H. Schenk and D. Bootsma

An experiment was carried out to determine whether the cells, which were in S phase at the m(unent of the addition of TdR to the cultures, are syn- chronized by a single treatment. In this experiment the procedure was as follows. After a labeling period of 20 rain with aH-TdR (0.5 #C per ml) in

100 -

g ' 90

g" 80

70

60-

50-

30-

20-

10-

/

Q.. 2 5 - - - r - ~ 5 - - , - - - , - - - ; - - - . - - . - , - - - , ~ = - -~

hours a/eel e~c~ ~Jt flr'~l thymi~ine treatment

22

20'

�9 ~ 1 8 �84

16-

12-

TO-

8 -

6 -

2-

Fig. 6.

"

haur~ aftQr qnd gf lhyrnidine troatmoat

Fig. 7.

Fig. 6 . - - P e r c e n t a g e of mi toses and labeled cells af ter a single t r e a t m e n t wi th 7.5 m M T d R . Label ing wi th a H - T d R was carr ied ou t i mmed i a t c l y pr ior to f ixat ion. O - - O , mi toses ; O - - O , labeled cells; , mi toses , m e a n in controls; . . . . , labeled cells, m e a n in controls .

Fig. 7 . - -Mi to t i c ac t iv i ty af ter single, double and tr iple t r e a t m e n t wi th 7.5 m M T d R . []--71, a l te r s ingle t r e a t m e n t ; O - - O , double t r e a t m e n t ; O - - O , tr iple t r e a t m e n t ; - - - , m e a n in controls .

control medium, the cultures were washed and incubated for 24 hr in 7.5 mM unlabeled TdR. Then the cells were washed three times with control medium and the percentage of labeled and total mitoses was determined at intervals. The results are presented in Fig. 5. The labeled mitoses represent cells that were in S phase at the beginning of the treatment, the unlabeled mitoses cells that were in G~, (;~ or M. Obviously the labeled fraction of the starting popu- lation was hardIy sync/n'onized at a l l

Fig. 6 shows that at 14 hr after a single treatment the percentage of cells in S phase was lowest, viz. about 5 per cent. Consequently this is the best moment to start the second treatment (see introduction). Comparison of the effect of double treatments, with various durations (12, 14, 16 or 18 hr) of

Experimental Cell Research 41

Sgnchronization bg inhibition of DNA sgnthesis. I 435

the first growth per iod, indeed showed opt imal sy n ch ro n y when the dura t ion was 14 hr. The tirst t rea tment was started when 26 per cent of the popula t ion was in S phase (Fig. 6, labeled cells, m e a n in controls) . The second t rea tment was started w h e n 5 per cent was in S phase. At about 14 hr after a double t rea tment the percentage of S phase cells again showed a m i n i m u m , at an even lower value, name ly at about 1 per cent [81. This means that a th i rd t rea tment could be effective and that the opt imal dura t ion for the second growth per iod shouhl also be 14 hr. In Fig. 7 the effects of single, double and triple t rea tments (Fig. 1) are compared .

In a p re l imina ry exper iment two de terminat ions of the plating efficiency after a t rea tment (see mater ia l and methods) were car r ied out. The results were for the plating efficiency at 6 hr after a single t rea tment 84 and 96 per cent of the controls and at 5.5 hr after a double t rea tment 71 and 98 per cent.

D I S C U S S I O / ~

The triple t rea tment (Fig. 1 C) was f lmnd to be the opt imal p rocedu re for synchron iza t ion by inhibi t ion of the DNA synthesis. The inocu lum is 6.10 ~ cells per Petri dish. The DNA synthesis is b locked bv the addi t ion of thymi- dine to the cul ture m e d i u m in a concent ra t ion of 7.5 lnM. The t rea tments last 24 hr. Deblocking of the 1)NA synthesis is achieved by washing the cells with control med inm. The dura t ion of the growth per iods between the t rea tments is 14 hr. The peak of the mitotic index in the synchron ized cul ture was fi)und to be about 12 times as high as the index in controls.

Applicat ion of a third t rea tment has the d isadvantage of compl icat ing con- s iderab ly the t ime scheme of exper iments with the synchron ized popula t ion . The double t rea tment p rocedu re will be m u c h more convenien t for rout ine exper iments . Theore t ica l ly more than three t rea tments could be used to br ing the percentage of S phase cells at the m o m e n t of the start of the last t rea tment down f rom 1 to 0 per cent. Since the inocululn could not be taken smaller than 6.10 a cells per Petri dish, the coverslips would get overcrowded, with all the concomi tan t disadvantages.

The plating efficiency of the synchron ized popula t ion was found to be only slightly lower than of un t rea ted controls. Qualitative microscopica l examina t ion showed no cel lular abnormal i t ies in the synchron ized cultures, c o m p a r e d with control populat ions . The incidence of certain types of mitotic aber ra t ions repor ted previous ly Ill was f imnd to be a character is t ic of the cell line used, ra ther than an effect of the synchroniz ing t reatments . Persistent

nucleoli as observed I111 after t rea tment with FUdB were not noticed here.

29- 661801 Experimenlal Cell Research 41

436 G. Galavazi, H. Schenk and D. Bootsma

Synchronization by treatment with TdR has also been obtained with other mammal ian cell lines, viz. monolayer cultures of Chang appendix [33] and HeLa [7, 24] cells anti Chinese hamster ovary cells in suspension I21]. The smaller the fraction of the total generation time occupied by the S phase, the better the synchrony that is to be expected after a single treatment. But a probably even more important requirement for good synchrony is the smallest possible variation of the generation time of the individual cells in the population.

The peak of the mitotic index after a single treatment with 7.5 mM TdR was found at 10 hr (in Figs. 2 and 3A) or at about 11 hr (in Figs. 4-7). Double treatment experiments with the same batches of cells showed peak values at respectively about 8.5 and 9.5 hr (only one curve is shown, see Fig. 7). This difference of 1 hr in the time of the peak was correlated with two distinct generation cycles as determined in random cultures of the respec- tive cell batches [2]. The characteristics of these two cycles were : G1, 12 hr; S, 8 hr; G2, 4 hr for the material of Figs. 2 and 3; and G1, 14 hr; S, 8 hr; G2, 5 hr for Figs. 4-7 (the duration of M has been divided between G1 and G~). This means that the differences in time of the peak can be correlated with equal differences in the duration of the G2 phase. Also it can be noticed that the peaks are higher in Figs. 2 and 3 A, where the percentage of mitoses in the controls is 2.8, than in Figs. 4-7, where the control percentage is 2.0-2.1. But even when the asynchronous percentage was equal, the height of the peak varied from one experiment to another (compare Figs. 2 and 3A). The finding that the time between the end of the synchronizing pro- cedure and the peak of mitotic activity was shorter after double than after single treatment and always shorter than the total duration of S and (12 will be discussed in the next paper [8], in which an analysis of some as- pects of the population dynamics before, during and after the synchro- nizing treatment will be presented.

S U M M A R Y

An optimal procedure to synchronize the life cycles of the individuals in a population of human cells, cultured in vitro, by repeated temporary in- hibition of the DNA synthesis was elaborated. Cultures in the logarithmic growth phase were treated three times with 7.5 mM thymidine dissolved in medium. Each treatment was terminated after 24 hr by washing the cultures with control medium. Between two successive treatments the cells were kept in the control medium for 14 hr. The peak of the mitotic index in the s y n -

Experimenlal Cell Research 41

S ! l t w h r o n i z , l ion by i n h i b i t i o n o [ ' D N A s y n t h e s i s . I 437

c h r o n i z e d c t d t u r e w a s f o t m ( I 1o be a b o u t 12 t i m e s as h i g h a s t h e i n d e x i n

c o n t r o l s . T h e n e c e s s i t y o f m o r e t h a n o n e t r e a t m e n t w a s d e m o n s t r a t e d .

S o m e p r o b l e m s c o n c e r n i n g m u l t i l ) l e t r e a t m e n t a r e d i s c u s s e d . A c l o n i n g

e x p e r i m e n t s h o w e d t h a t o n l y l i t t le i r r e v e r s i b l e d a m a g e is c a u s e d b y t h e s w l -

c h r o n i z i n g h ' e a t m e n l ; m i c r o s c o p i c a l l y n o c e l l u l a r a b n o r m a l i t i e s w e r e o b -

s e r v e d .

The authors wish to express their gratitude to Dr O. Vos for m a n y helpful discus- sions.

REFERENCES

1. BOOTSMA, 1)., BUD~ZE, I,. and Vos, O., E.rpll Cell Ices. 33, 301 (1964). 2. BOOTSMA, 1)., I(AALI';N, 31. C. A. C. and GALAVAZI, G., In preparation. 3. BnEwnx, J. G., (;enelics 50, 101 (1964). 4. I)UNCAN, 1/. }'~. and \\'OODS, P. S., Chromosoma 6, 15 (1953). 5. EIDINOFF, ~[. lJ. ~111(| [~ICH, .~1. A., Cancer /~es. 19, 521 (1959). 6. ]~RIKSON, 1/. L. and SZS'BALSKI, \V., I~adialion lies. 18, 200 (1963). 7. FIRKI.iT, H., Colllpl. Icend. Soc. Biol. 158, 141)8 (1961). 8. GALAVAZI, G. an(l BOOTSMA. D., Expll (;ell lles. 41, 438 (1966). 9. GOLD, M. and l~h'LLE1X~.:n, C. \V., Biochim. llioph!ls. Acla 80, 193 (1964).

10. I-tEII)ELI~EI~.GEH, (]., Expll Cell Ices. Suppl. 9,462 (1963). 11. Hsu, T. C., Hum,m~l;Y, 11. 3I. and SoM~ns, C. S., Expll (Jell Res. 33, 74 (1964). 12. HUrNNEKI,:NS, F. M., BEnTt:<O, .1. 11., SILBEn, R. and GABnIO, B. W., Expll Cell lees. Suppl. 9,

441 (1963). 13. KA.nWAI~A, K. and MUELLEn, G. C., Fed. Proc. 21, 163 (1962). 14. - --- Iciochim. Bioph!ls. Aela 91, 486 (1964). 15. I'(LENOW, 11., Biochim. IHophys. Acla 61, 885 (1962). 16. LITTLEFIELI), .J.W., Expll (:ell ICes. 26, 318 (1962). 17. Mom~is, N. 1~. and I"ISCIIER, ('~. A., Ir Biophys. Acla 68, 84 (1963). 18. Monms, N. R., REWH.~,m), P. an(l lqscm.:n, G. A., Biochim. Biophgs. Aela 68, 93 (1963). 19. MUELLEn, G. C., Expll Cell ICes. Suppl. 9, 144 (1963). 2t). P.cIY'rEn, t~. B. and I~.AS.MUSSE>,', 1/. 1'~., Nalttre 201, 409 (1964). 21. PETI.:nsEN, D. F. and ANDVnSOX, E. C., Nature 203, 642 (1964). 22. PnENSKY, W. and SMITII, H. H., Expll ('ell lies. 34, 525 (1964). 23. PUCK, T. T., .I. (:ell Itiol. 19, 57 A (1963). 2-1. ------ Science 144, 565 (1961). 25. I'~UECKEI{T, l:{. |{. and ~IUELLEI{, G. (]., Cam'er Res. 20, 1584 (1960). 26. SCrIACWrs('nA~EL, D. an(I MAZZ()NE, It. M., Pro('. Am. Assoc. Cancer Ices. 5, 56 (196-1). 27. SCnINDLEn, R., 13iochem. Pharmae~,l. 12, 533 (1963). 28. SMITH, H. 1t., I'USSELL, C. P. and KU(;ELr~AN, B. It., Science 142, 595 (1963). 29. TILL. J. E., \VnITMOnE, G. F. and GULYAS, G., 13iochim. Biophgs. Acla 72, 277 (1963). 30. VEEN, .I. X'~X l)ZR, BOTS, 1,. and Mns, A., Arch. Virus[orsch. 8, 230 (1958). 31. Vos, O., BUDrCE, 1~. and V~nGnOESEN, A. J., lnlern.. l . Icadialion Biol. !5, 543 (1962). 32. Vos, O. and KAALEN, M. C. A. (]., ibid. 5, 6119 (1962). 33. XEn()S, N., Nature 194, 682 (1962).

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