Studies on DNA Replication in Animal Cells (Annual Report) EUR 2959

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EUROPEAN ATOMIC ENERGY COMMUNITY — EURATOM

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DIES ON DNA REPLICATION IN ANIMAL CELLS

(Annual Report)

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, Ν. DUTHEILLET-LAMONTHEZIE, Ph. JEANTEUR

and A. OBRENOVITCH

(Institut Gustave Roussy)

1966

Report prepared by the Biochemistry and Enzymology Laboratory

Institut Gustave Roussy — Villejuif, France

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E U R 2959. e

STUDIES ON DNA REPLICATION IN ANIMAL CELLS (Annual Report) b y C. PAOLETTI, N. DUTHEILLET-LAMONTHEZIE, Ph. J E A N T E U R and A. OBRENOVITCH (Institut Gustave Roussy).

European Atomic Energy Community — EURATOM

Report prepared by the Biochemistry and Enzymology Laboratory, Institut Gustave Roussy, Villejuif (France)

Euratom Contrat No. 042-64-10 B I O F

Brussels, August 1966 — 18 pages — 6 figures — FB 40

Ehrlich and Krebs ascites cells DNA is pulse labelled by 8H-thymidine

in vivo and extracted at various times between 5 minutes and 7 hours after this pulse; radioactive material of the 5 min. DNA displays, when compared to bulk DNA, a higher affinity to MAK columns, a slower sedimentation rate and an increased sensitivity to alkaline denaturation without any change in CsCl and Cs2S04 buoyant density.

In DNA extracted longer after this pulse, the p a r t of radioactive material which shows u p such characteristics becomes progressively less important and in 2 hours DNA the behaviour of labelled material and bulk DNA is nearly identical. The best explanation of these data is the existence of structurally modified DNA, correlated to its replication.

E U R 2959. e

STUDIES ON DNA REPLICATION I N ANIMAL CELLS (Annual Report) b y C. PAOLETTI, N. D U T H E I L L E T - L A M O N T H E Z I E , Ph. J E A N T E U R and A. OBRENOVITCH (Institut Gustave Roussy).

European Atomic Energy Community — EURATOM

Report prepared b y the Biochemistry and Enzymology Laboratory, Institut Gustave Roussy, Villejuif (France)

Euratom Contrat No. 042-64-10 B I O F

Brussels, August 1966 — 18 pages — 6 figures — FB 40

Ehrlich and Krebs ascites cells DNA is pulse labelled by sH-thymidine

in vivo and extracted at various times between 5 minutes and 7 hours after this pulse; radioactive material of the 5 min. DNA displays, when compared to bulk DNA, a higher affinity to MAK columns, a slower sedimentation rate and an increased sensitivity to alkaline denaturation without any change in CsCl and Cs2S04 buoyant density.

In DNA extracted longer after this pulse, the p a r t of radioactive material which shows u p such characteristics becomes progressively less important and in 2 hours DNA the behaviour of labelled material and bulk DNA is nearly identical. The best explanation of these data is the existence of structurally modified DNA, correlated to its replication.

E U R 2959. e

STUDIES ON DNA REPLICATION IN ANIMAL CELLS (Annual Report) b y C. PAOLETTI, N. D U T H E I L L E T - L A M O N T H E Z I E , Ph. J E A N T E U R and A. OBRENOVITCH (Institut Gustave Roussy).

European Atomic Energy Community — EURATOM

Report prepared by the Biochemistry and Enzymology Laboratory, Institut Gustave Roussy, Villejuif (France)

Euratom Contrat No. 042-64-10 B I O F

Brussels, August 1966 — 18 pages — 6 figures — F B 40

Ehrlich and Krebs ascites cells DNA is pulse labelled b y 'H-thymidine

in vivo and extracted at various times between 5 minutes and 7 hours after this pulse ; radioactive material of the 5 min. DNA displays, when compared to bulk DNA, a higher affinity to MAK columns, a slower sedimentation rate and an increased sensitivity to alkaline denaturation without any change in CsCl and Cs2S04 buoyant density.

EUR 2359.e

EUROPEAN ATOMIC ENERGY COMMUNITY — EURATOM

STUDIES ON DNA REPLICATION IN ANIMAL CELLS

(Annual Report)

by

C. PAOLETTI, N. DUTHEILLET-LAMONTHEZIE, Ph. JEANTEUR

and A. ORRENOVITCH

(Institut Gustave Roussy)

1966

Report prepared by the Biochemistry and Enzymology Laboratory Institut Gustave Roussy — Villejuif, France

SUMMARY

Ehrlich and Krebs ascites cells DNA is pulse labelled by 3H-thymidine

in vivo and extracted at various times between 5 minutes and 7 hours after this pulse; radioactive material of the 5 min. DNA displays, when compared to bulk DNA, a higher affinity to MAK columns, a slower sedimentation rate and an increased sensitivity to alkaline denaturation without any change in CsCl and Cs2S04 buoyant density.

CONTENTS

1 — INTRODUCTION 5

2 — MATERIALS AND METHODS 5

2.1 — Chemicals 5

2.2 — Counting of radioisotopes 6

2.3 — Biological material and technique of labelling 6

2.4 — DNA extraction 6

2.5 — Methylated albumin column chromatography 7

2.6 — Sucrose gradients 7

2.7 — Buoyant density gradients 8

2.8 — Miscellaneous techniques 8

3 — RESULTS 8

3.1 — Analytical properties of DNA and efficiency of extraction procedure 8

3.2 — Chromatographic behaviour of 5 min. DNA and 7 hrs DNA 9

3.3 — Behaviour of radioactivity of 5 min. and 7 hrs DNA in sucrose gradient

centrifugation 11

3.4 — Behaviour of radioactivity of 5 min. and 7 hrs DNA in density gradients 12

3.5 — Increased sensitivity to alkaline denaturation of radioactive material

of 5 min. DNA 13

3.6 — Kinetic data 14

4 — DISCUSSION 16

REFERENCES 17

TABLE I Distribution of UV absorbing material and radioactivity among the three

chromatographic fractions and the NE part of 5 min. DNA 10

TABLE II Distribution of radioactivity in the different chromatographic fractions

and the NE part in 5 min. DNA's before and after CsCl centrifugation

K E Y TO T H E FIGURES

FIG. 1 — Methylated albumin chromatography with gradient elution of 5 min and 7 hrs

DNA's.

F I G . 2 — Sucrose gradient centrifugation (15-5 %) of radioactive DNA.

FIG. 3 —■ Neutral CsCl density gradient of a five min. DNA — Total number of fractions

196 with a density gradient of about 0,001 g/cm

between two drops.

FIG. 4 — Density gradient centrifugation in CsCl at alkaline p H .

FIG. 5 — Specific activities of total DNA's extracted by the Schmidt and Thannhauser

procedure at various times after thymidine —

H injection (25 μο/πιοιιββ) and

chase by 2 μΜ of cold thymidine 5 min. after the label injection.

For each time, ascites cells from 15 mice Avere pooled.

STUDIES ON DNA REPLICATION IN ANIMAL CELLS (*)

1 — INTRODUCTION

The semi-conservative scheme of DNA rejdication has been demonstrated for

bacteria (Meselson and Stahl, 1958) and extended to mammalian cells (Simon, 1961); it

implies the existence of an intermediate structure between those of the mother and the

two daughters molecules in which each parental single-strand is bound to both parental

and daugther complementary single-strands; such a structure may be called replicative

form of DNA; it has been inferred to have a Y like shaped structure associated with some

specific physico-chemical properties.

Autoradiographic (Cairns, 1963), genetic (Yoshikawa and Sueoka, 1963), biophysical

(Baldwin and Shooter, 1963; Rolfe, 1963; Hanawalt and Ray, 1964; Lark and Lark, 1964;

Rosenberg and Cavalieri, 1964; Guérineau, Dechambre and Paoletti, 1966) data have

been accumulated in support of such a scheme for bacteria. DNA replicative forms have

also been demonstrated into bacterial viruses (Frankel, 1963; Kozinski and Lin, 1965;

Smith and Skalka, 1966). In animal systems, data are more scanty (Ben Porat, Steere

and Kaplan, 1962; Rosenberg and Cavalieri, 1964, Cairns, 1966). Nevertheless, the existence

of replicative forms in DNA may be reasonably assumed. The present paper brings some

experimental arguments in favour of this hypothesis.

The basis of the experimentation was as following : mouse Ehrlich ascites cells Avere

given a pulse of a radioactive precursor of DNA (

H thymidine) ; the label of a DNA

extracted soon after this pulse must be found in molecules (newly labelled DNA) which

had just been synthesized. Would replicative forms exist, they should be labelled at that

time. On the other hand, when DNA was extracted later on, the label must be into

mole-cules synthesized since a long time and consequently any eventual replicative form would

not be labelled.

In case these forms have some specific features, the behaviour of radioactive material

from each DNA should be different when subjected to technics which differentiate DNA's

according to their molecular size, shape or structure, such as chromatography on methylated

albumine, zone centrifugation in sucrose, buoyant density centrifugation in CsCl and

Cs

S0

, and alkaline denaturation. The results displayed such differences and Avere in support

of the existence in animal cells of transient macro-molecular forms of DNA related to its

duplication.

2

—

MATERIALS AND METHODS

2.1

—

Chemicals

Sucrose was an Anular product. Bovine albumin (fraction V) was obtained from

Armour Company. Non radioactive thymidine and venom phosphodiesterase Avere Sigma

Chemical products. CsCl was purchased from American Potash Chemicals and

recrystal-lised before use. Cs

SO, was a Merck product. Trypsin, chymotrypsin, pancreatic DNasc

(*) Muitu*cri|>t vwpivml o« Janwarv H, l%t>.

and R N a s c Avere all p r o d u c t s of W o r t h i n g t o n Biochemical C o r p o r a t i o n . R N a s e w a s always boiled t e n m i n u t e s in a c e t a t e buffer 0.15 M, p H 5 before u s e .

T h y m i d i n e m e t h y l 3H (6.7 C/mM) Avas p u r c h a s e d from NCAV E n g l a n d Nuclear Corp.

a n d t e s t e d for radiochemical p u r i t y b y p a p e r c h r o m a t o g r a p h y before use (90 % of (:iH)

Avere Avith t h y m i d i n e s p o t ) .

2.2 — Counting of radioisotopes

D e t e r m i n a t i o n s of (3H) Avere done in a T r i c a r b Liquid Scintillation S p e c t r o m e t e r .

A q u e o u s s a m p l e s of 0.5 ml t o 1 m l were p u t on K l e e n e x p a p e r s (8 cm X 3 cm) f i t t e d to t h e inside Avail of c o u n t i n g Arials. After d r y i n g , t h e p a p e r s Avere i m p r e g n a t e d b y 0.8 ml

of a scintillation solution : 4 g of 2­5 d i p h e n y l o x a z o l e a n d 100 mg of 1.4­bis­2­(4 m c t h y l ­ 5­phenyloxazolyI b e n z e n e dissolved in 1 litre t o l u e n e . Efficiency of c o u n t i n g was 20 % .

2.3 — Biological material and technique of labelling

E h r l i c h a d e n o c a r c i n o m a cells r e g u l a r l y t r a n s p l a n t e d into the p e r i t o n e u m of C57

black mice h o m o g e n e o u s w i t h r e s p e c t t o age a n d sex h a v e been used t h r o u g h o u t t h i s Avork. F o u r or five d a y s after t h e graft of 25 X 106 cells, t h e mice Avere i n t r a p e r i t o n e a l l y given

25 μc of (3H) t h y m i d i n e in 1 ml of physiological saline u n d e r sterile conditions. K r e b s ­ 2

ascites cells h a v e also been u s e d . T h e y Avere t r a n s p l a n t e d into t h e p e r i t o n e u m of inbred SAVÌSS mice. T h e a n i m a l s Avere injected w i t h 3H t h y m i d i n e seven d a y s after t h e graft of

20.106 cells. A n i m a l s Avere killed tAvo m i n u t e s before t h e i n d i c a t e d t i m e of labelling a n d t h e

ascitic fluid AvithdraAvn from t h e p e r i t o n e u m i n t o a t u b e c o n t a i n i n g a fcAv m l of SC (*) at 37° C. A t t h e i n d i c a t e d t i m e , t h e t u b e s Avere frozen at — 80° C. 5 m i n u t e s Avas chosen as a t i m e c o m p a t i b l e Avith an easy h a n d l i n g of mice a n d o b t e n t i o n of a newly labelled D N A of a sufficiently high specific actiA'ity for a c c u r a t e c o u n t i n g ; 7 h o u r s was chosen in conside­ r a t i o n of t h e cellular a n d mitotic cycle of E h r l i c h ascites cells (EdAvards, 1960) to rule out t h e possibility t h a t D N A s y n t h e s i z e d d u r i n g t h e pulse Avould e n t e r into a n e w replication process at t h e e x t r a c t i o n t i m e ; i n t e r m e d i a t e t i m e s Avere selected t o o b t a i n kinetic d a t a .

T h e d i s a p p e a r a n c e ef r a d i o a c t i v i t y from t h e p e r i t o n e a l caA'ity is A'ery r a p i d : after 2 m i n u t e s , 25 % of t h e injected t r i t i u m Avas recoAcred in t h e ascitic fluid a n d only 13 % after 5 m i n u t e s . Consequently, one c a n consider t h a t a physiological pulse; a c t u a l l y took place. N e v e r t h e l e s s , in k i n e t i c e x p e r i m e n t s , 2 μ moles of unlabelled t h y m i d i n e Avere given 5 m i n u t e s after a d m i n i s t r a t i o n of t h e label.

P l e u r o p n e u m o n i a ­ l i k e organisms could n o t be found in forteen different ascites cells s a m p l e s , except for one case (Klieneberger.­Nobel E . , 1962). B a c t e r i a Avere n e v e r found.

2.4 — DNA extraction

Labelled D N A Avas p r e p a r e d according to the p r o c e d u r e of D u t h e i l l e t ­ L a m o n t h é z i e a n d G u é r i n e a u (1965). F r o z e n cells stored at — 80° C Avere t h a w e d at 0° C, washed three­ t i m e s Avith SC a n d b r o k e n i n SC b y u l t r a ­ T u r r a x (3 seconds) or teflon­pestled P o t t e r .

(*) AbreA'iations :

MAK : methylated albumin coated Kieselguhr.

SC : saline citrate; 0.14 M NaCI containing 0.05 M sodium citrate. 1/10 SC : SC diluted tenfold.

5 min DNA and 7 h DNA refer to DNA's extracted from cell* withdiawu ."> minutes and 7 hours after label injection.

SA : specific activity expressed as cts/min/OD unit.

dAT : double stranded copolymer of A and Τ deoxyribonucleotides.

Centrifugation 15 m i n u t e s a t 30,000 g was r u n t o r e m o v e t h e b u l k of c y t o p l a s m i c R N A Avhile t h e pellets Avere essentially m a d e u p of nuclei. These pellets Avere w a s h e d a n d ccntri­ fuged t h r e e t i m e s before b e i n g r e s u s p e n d e d b y u l t r a ­ T u r r a x (3 seconds) or P o t t e r in a sufficient a m o u n t of SC t o h a v e a final D N A c o n c e n t r a t i o n of 400 t o 800 μg/ml. P h e n o l solution (phenol : 9 v o l u m e s ; E D T A 1 0 "3 M : 1 v o l u m e ; c o n c e n t r a t e d K O H u p to p H 9.0)

was sloAvly a d d e d t o the suspension Avith s h a k i n g at r o o m t e m p e r a t u r e . After one h o u r s h a k i n g , t h e viscous, m i l k y suspension Avas centrifuged for 30 m i n u t e s at 30,000 g. T h e u p p e r a q u e o u s l a y e r Avas t h e n carefully withdraAvn a n d subjected t o a second p h e n o l t r e a t ­ m e n t d u r i n g 15 m i n u t e s . After centrifugation as a b o v e , 10 v o l u m e s of t h e a q u e o u s layer c o n t a i n i n g D N A received one v o l u m e of 1.5 M s o d i u m a c e t a t e buffer p H 5.5. P h e n o l Avas t h e n e x t r a c t e d b y s h a k i n g t e n t i m e s w i t h t h r e e v o l u m e s of p e r o x i d e free e t h e r containing acetic acid (1/750 V/V). E t h e r Avas finally r e m o v e d b y n i t r o g e n b u b b l i n g .

T h e l a s t tAvo steps Avere R N a s e t r e a t m e n t (10 ¡.¿g/ml) according t o Rolfe (1963) and e t h a n o l p r e c i p i t a t i o n b y a d d i n g tAvo v o l u m e s of e t h a n o l s a t u r a t e d w i t h p o t a s s i u m a c e t a t e folloAved b y redissolution in 1/10 SC. T h e l a s t s t e p s Avere often p u r p o s e l y o m i t t e d because neAvly labelled D N A molecules m i g h t be s u p p o s e d to h a v e such a s t r u c t u r e as t o be modified b y e t h a n o l p r e c i p i t a t i o n .

S o m e t i m e s , p r o t e o l y t i c e n z y m e s p r i o r t o p h e n o l e x t r a c t i o n Avere used. T h e pellet c o n t a i n i n g nuclei Avas lyscd b y t r e a t m e n t w i t h a 6 M NaCI solution Avhich r e s u l t e d in a d r a m a t i c increase of viscosity. T h e saline c o n c e n t r a t i o n Avas loAvered b y dialysis against SC t o a b o u t 0.2 M. One half of t h e solution Avas e x t r a c t e d b y p h e n o l a n d t h e o t h e r one t r e a t e d b y c h y m o t r y p s i n (10 [j.g/ml) a n d t r y p s i n (10 μg/ml) for t h r e e h o u r s at 37° C, before p h e n o l t r e a t m e n t .

2.5 — Methylated albumin column chromatography

TAVO m e t h o d s Avere c o n c o m i t a n t l y used :

2.5.1 —· Continuous elution m e t h o d . T h e e x p e r i m e n t a l p r o c e d u r e of Mandell a n d H e r s h e y (1960) Avas followed, t h e a m o u n t of t h e t h r e e layers being h a hre d . T h e Avater­

j a c k e t t e d c o l u m n (1.8 cm X 12 cm) Avas l o a d e d Avith 10 t o 21 O D u n i t s of e x t r a c t e d m a t e r i a l ( a b o u t 0.4 OD u n i t / m l ) in 0.4 M NaCI 0.05 M p H 6.8 p h o s p h a t e buffer. An e x p o n e n t i a l NaCI g r a d i e n t betAveen 0.4 a n d 1.0 M in 0.05 M p H 6.8 p h o s p h a t e buffer Avas first applied folloAved b y a p H g r a d i e n t in 1.0 M NaCI u p t o p H 10.5 w i t h 1.0 M NaCI 1.5 N N H4 O H

solution (Lacks, 1962). NaCI c o n c e n t r a t i o n of t h e g r a d i e n t Avas m e a s u r e d b y r e f r a c t o m e t r y .

2.5.2 — StepAvise elution p r o c e d u r e as described b y Sucoka a n d Cheng (1962a). T h e s a m e a m o u n t s of m a t e r i a l as aboA'e Avere l o a d e d in 0.5 M NaCI — 0.05 M p H 6.8 phos­ p h a t e buffer a n d eluted b y 0.1 M increase of s a l t c o n c e n t r a t i o n in 0.05 M p H 6.8 p h o s p h a t e buffer; for each s t e p , four 5 m l fractions Avere collected. A d d i t i o n a l elution step Avas done b y 20 m l of 1.0 M NaCI — 1,5 M N H4 O H p H 1G.5 solution.

I n b o t h c h r o m a t o g r a p h i c p r o c e d u r e s , t e m p e r a t u r e Avas k e p t c o n s t a n t a t 20 + 2° C.

2.6 — Sucrose gradients

2.7 — Buoyant density gradients

2.7.1 — At n e u t r a l p H , CsCl s t o c k solution was 62 % ( W / W ) in distilled w a t e r . Labelled m a t e r i a l (1 t o 5 OD units) in n e u t r a l buffer or SC solution was a d d e d along to m a k e density a r o u n d 1,690 g/ml 3 ml of such a solution were r u n in t h e S W 39 r o t o r of a L2 Spinco p r e p a r a t i v e U l t r a c e n t r i f u g e at 25° C for 50 hours at 35,000 r c v / m i n One or tAvo drop fractions Avere collected after piercing t h e b o t t o m of t h e t u b e , diluted Avith 1 m l of distilled Aväter a n d a s s a y e d for O D a n d radioactiArity.

Cs2S04 stock solution Avas 45 % ( W / W ) in distilled Aväter. Labelled m a t e r i a l solution

(1 t o 5 O D units) a n d saline Avere a d d e d along u p t o a d e n s i t y a r o u n d 1,430 g/ml 2.5 ml of such a solution Avere r u n for 40 h o u r s at 31,000 r e v / m i n as above and dropwise collected. Densities Avere deriA'cd from refractÎA'e i n d e x .

2.7.2 — A t alkaline p H r e p r o d u c i b l e p H m e a s u r e m e n t s could be o b t a i n e d using a r a d i o m e t e r P H M 4 C p H m e t e r s t a n d a r d i z e d Avith s a t u r a t e d Ca(OH)2 solution prepared

j u s t before use in freshly distilled Aväter a n d filtered t h r o u g h a sin.tercd­glass, p H of t h i s solution was 12.45 a t 25° C a n d all d e t e r m i n a t i o n s Avere performed in a Avatcr b a t h at c o n s t a n t t e m p e r a t u r e (25° C). To 3 ml of CsCl stock solution Avere a d d e d 0.2 ml of t h e e x t r a c t e d m a t e r i a l solution (1 t o 5 OD units) in 0.14 M NaCI t h e n 0.3 ml of 0.4 M p o t a s s i u m p h o s p h a t e buffer p H 10.95. S a t u r a t e d K O H solut­on, diluted t o 1/50 before use was added Avith a m i c r o p i p e t t e u p t o p H r a n g i n g b e t w e e n 11.35 and 11.48. Centrifugations Avere as a b o v e for 50 h o u r s .

2.8 — Miscellaneous techniques

O D m e a s u r e m e n t s Avere done Avith a Zeiss PM(^ I I s p e c t r o p h o t o m e t e r . Dosage of' nucleic acid c o m p o n e n t s in cells Avas performed according t o t h e S c h m i d t and T h a n n ­ h a u s e r (1945) p r o c e d u r e . D N A a n d R N A c o n t e n t s Avere m e a s u r e d by d i p h e n y l a m i n c reaction for deoxyribose ( B u r t o n , 1956) a n d orcinol reaction for ribosc (Moule, 1953). Alkaline t r e a t m e n t of phenol e x t r a c t e d D N A or c h r o m a t o g r a p h i c fractions Avas mad«; b y 0.5 N K O I I 16 hours at 37° C. P r o t e i n d e t e r m i n a t i o n s Avere performed after exhaustÏA e dialysis of phenol Avith t h e procedure of LoAvry, R o s e n b r o u g h . F a r r a n d R a n d a l l (1951), bovine a l b u m i n being t a k e n as a s t a n d a r d . D N A s e t r e a t m e n t Avas performed b y 10 μg/ml e n z y m e at p H 7.5 a n d 10~2 M. M g+ + 1 h o u r at 37° C. R N a s e t r e a t m e n t Avas accomplished according to Rolfe

(1963).

Electrophoresis of nucleotides Avas performed Avith the a p p a r a t u s described !>\ M a r k h a m and S m i t h (1952) in 0.02 M s o d i u m c i t r a t e buffer p H 3.5. Ribonucleotides were o b t a i n e d b y alkaline hydrolysis (cf aboAe) and deoxynucleotides b y e n z y m a t i c hydrolysis with p a n c r e a t i c D N a s e a n d v e n o m phosphodiesterase. T h e t e c h n i q u e of W y a t t (1951) Avas used for p a p e r c h r o m a t o g r a p h y of radioactiAe t h y m i d i n e and bases o b t a i n e d 1>A per­ chloric hydrolysis of D N A .

B o u n d a r y s e d i m e n t a t i o n m e a s u r e m e n t s Avere carried out in t h e 12 m m . Kel­F center­ piece of a Spinco model E a n a l y t i c a l ultracentrifuge at 44,770 r e v / m i n at 20° C Avith 0.2 O D u n i t / m l , a c o n c e n t r a t i o n small enough to alloAV one t o neglect t h e c o n c e n t r a t i o n effect.

3 — RESULTS

3.1 — Analytical properties of DNA and efficiency of extraction procedure

T h e yield of phenol e x t r a c t e d D N A , c o m p a r e d to the D N A c o n t e n t e v a l u a t e d b A' t h e Schmidt and T h a n n h a u s e r (1945) p r o c e d u r e Avas close t o 100 % . T h e relative yield of (3H) cts (compared t o yield of U.V. absorbing material) gave quite different results with

5 min and 7 hrs D N A ' s : 40 to 70 % of the total radioactivity of 5 min. D N A was rccoAcred by pinino! extraction while nearly 100 % Avas obtained with 7 h D N A . UnrecoA'ered radio-activity of 5 min D N A Avas localized in the aqueous phenolic interfacial layer from Avhich it could be partially recovered b y further t r e a t m e n t Avith proteolytic enzymes and phenol; it has already been reported t h a t « nascent » D N A molecules are more resistant to extrac-tion (Goldstein and BroAvn, 1961; Ben P o r a t , Steere and K a p l a n , 1962).

When D N A preparations were analysed prior to RNasc t r e a t m e n t and ethanol precipitation, they contained some R N A a m o u n t i n g to 20-25 % and 2-5 % proteins. Otherwise, they contained less than 2 % of R N A or proteins.

Sedimentation coefficients observed with different lots of DNA were between 24 and 32 S. No difference betAvcen ultra-Turrax and hand-teflon-pestlcd P o t t e r extracted D N A ' s was observed.

3.2 — Chromatographic behaviour of 5 min. DNA and 7 hrs DNA

3.2.1 - - Definition of fractions :

Three fractions Avere conventionally defined :

— 0.7 M fraction : bulk of D N A eluted before 0.8 M NaCI elution.

- 0.8 M fraction : material eluted betAveen 0.8 M and 1.0 M NaCI at p i I 6.8. — p H fraction : material eluted during p H increase at 1.0 M NaCI.

Moreover, a p a r t of radioactiAe material cannot be eluted from t h e column, either under the above conditions or b y N a O H N, HCl N, 5 M NaCI, proteolytic enzymes, Na dodecyl sulfate 0.5 % in NaCI 0.01 M; it has been called N E (non elutable) part of radioactive DINA. Since then, this radioactive p a r t has been eluted from the column by a 0.1 M P 04N a3

solution.

Some U.V. absorbing material, presumably hydrolyzed or S - R N A Avas not adsorbed on MAK columns, it did not contain any significant a m o u n t of radioactivity.

3.2.2 — Elution patterns of 5 min and 7 h DNA (Fig. 1).

With 7 h D N A , profiles of U.V. absorbancy and radioactivity obtained by both elution procedures Avere closely superimposed, indicating a homogeneous distribution of labelled molecules in all fractions. On the contrary, 5 min DNA exhibited quite different p a t t e r n s of radioactiA ity and U.V. absorbancy and displayed two remarkable

characteris-tics : (a) the so-called 0.8 M and p H fractions shoAved a much higher specific actiA ity than

0.7 M one; (b) the N E p a r t is relatiA'ely i m p o r t a n t . QuantitatiA e evaluation is given in Table I. Earlier chromatographic fractions eluted near 0.6 M NlaCI are enriched up to 20 % in satellite b a n d as described by Cheng and Sueoka (1963).

E A 'idence t h a t all the radioactive label Avas associated Avith a deoxyribonucleic material was obtained :

— When 5 min D N A Avas subjected before c h r o m a t o g r a p h y to DNasc degradation, less t h a n 5 % of radioactiA ity Avas adsorbed on MAK columns and eluted all over salt gradient.

TABLE I

Distribution of UV absorbing material and radioactivity among the three chromatographic fractions and the NE part of S min DNA

Stepwise elution (*)

OD (3H)

Continuous elution (**)

O l ) (3H)

0.7 M fraction 0.8 M fraction ρ II fraction NE part . . .

75 ± 7 14 ± 4

9 + 4 —

30 ± 8 26 ± 5 15 -!- 3

29 ± 10

78 11 10 — za 35 2(1 2(1

Expressed as percentage of OD units or radioactivity adsorbed on the MAK columns.

-ihx — mf

(*) 19 experiments; σ = 1/ ­ f η — 1 (**) 7 experiments ­ arithmetical mean.

SALT G R A D I E N T pH GRADIENT

0.8

Q < _

t

Q4

0.2

0.8 M

Q 6 M

O 1

-Ο,β

- 0 /

t t t ì t , t t f c É i f c f c A A^ ^ ^k A A A A A á l

£2a

0,6 M

j a t a

Ο,βΜ

η - ^ ν ^ 1 > . η . . ^ . . η . . η - . ^ . .π ^ » r , - t . ^ > n . r , . . n ^ . A

0,75

_ 0,5

0,25

o - o '

-°.<

A 2

9 0 120 150 1 8 0 210

etriuenl ml

2 4 0 25 50

-°-«-°-*-"«°*·"

10

75

FlG. 1 — Methylated albumin chromatography with gradient elution of 5 min and 7 h DNA's.

a) 5 min DNA : 19.1 OD units, 81,900 cts/min Recovery : OD : 101 % ; (3H)cts : 82 % .

b) 7 h DNA : 10.8 OD units, 286,280 cts/min Recovery : OD : 1 0 4 % ; (3H) cts : 95 % .

OD — A — A — A — ; ( Ή ) - - o - - o - -.

— Existence of radioactive RNA Avas disproved by the folloAving data. Neither c h r o m a t o ­ graphic p a t t e r n s nor banding in CsCl Avere affected b y RNasc (10 μg/ml). 95 % or more of t h e radioactivity remained in the acid precipitable form after alkalini; t r e a t m e n t of total and 0.8 M fraction of 5 min D N A ; the small a m o u n t of alkali-sensible material in 5 min D N A could not be found in 7 h D N A , and after electrophoresis together Avith alkali-hydrolyzcd RNA yielded no radioactivity associated with usual ribonucleo­ tides (not even with ribothymidylic acid). P a p e r c h r o m a t o g r a p h y of perchloric hydro-lysate of total DNA alloAved 90-95 % of (:3H) counts to be recovered on t h y m i n e

spot-Likewise, 90-95 % of radioactivity was found associated with thymidylic acid after electrophoresis of enzymatic hydrolysate of 5 min D N A .

Three main criteria arc, involved in D N A fractionation by MAK column : G-C content, molecular weight and extents of hydrogen bonding (Sueoka and Cheng, 1962a). Fractionation according only to a difference in base composition cannot explain our results because a nearly pure d A T (crab satellite DNA) is eluted before 0.8 M NaCI (Sueoka and Cheng, 1962 b) while most of t h e radioactive material concerned here is still adsorbed at this molarity. F u r t h e r m o r e , based on the assumption of a m a x i m u m enrichment (100 %) in Α-T base pairs, the specific activity of radioactive fractions could not exceed 1.7 times t h a t of bulk DNA (1.7 being t h e ratio of 100 % to 58 % Α-T content of mouse DNA). Higher ratios actually were observed.

3.2.3 — Influence of protein contamination

CsCl centrifugation eliminates the most i m p o r t a n t p a r t of proteins remaining Avith D N A after extraction. Since radioactive material, in a prcA iously centrifuged D N A , shoAved chromatographic diagrams similar to those obtained with non centrifuged D N A , proteins could not explain t h e high affinity of this material for MAK. Nevertheless, the existence of small a m o u n t s of proteins strongly bound to D N A could not be ruled out and Avas demon­ strated for phage P j transducing particles D N A (Ikeda and TomizaAva, 1965).

Consequenth', the effect of trypsin-chymotrypsin on a 5 min D N A was tested, t h e enzA'mes being remoA ed by a phenol t r e a t m e n t . In this case also, no significant modi­ fication Avas obserAed Avhen chromatographic diagrams before and after t r e a t m e n t Avere compared (Tabic II).

TABLE II

Distribution of radioactivity in the different chromatographic fractions and the NE part in 5 min DNA's before and after CsCl centrifugation or enzyme treatment.

CsCl centrifugation Enzymes treatment

0.7 fraction 0.8 fraction Ρ fraction NE part . .

32.4 25.0 18.2 24.4 37.2 29.7 10.7 22.4 12.7 20.0 11.0 56.3 12.3 26.7 13.5 47.5

The results are expressed in percentage of radioactivity adsorbed on the MAK columns.

The centrifuged DNA and enzyme treated DNA were extracted from two different lots of animals.

3.3 — Behaviour of radioactivity of 5 min and 7 h DNA in sucrose gradient centrifugation

Similar results Avere obtained when 10 μg or 200 μg of' DINA Avere layered on top of sucrose gradients. Consequently, Ave generally used 200 or 300 μg of DNA to avoid too IOAV U.V. absorbancy. In a 7 h D N A , U.V. material and radioactivity (Fig. 2b) showed

t h e s a m e r a t e of s e d i m e n t a t i o n while, in a 5 min D N A (Fig. 2α), the r a d i o a c t i v i t y s e d i m e n t e d more slowly t h a n t h e U.V. m a t e r i a l . R N A s e p r e t r e a t m e n t or alcohol precipitation did n o t modify significantly t h e centrifugation p a t t e r n s .

Difference in molecular size alone could n o t explain these r e s u l t s ; if r a d i o a c t i v e m a t e r i a l differed only b y a loAver molecular size from b u l k D N A , it should h a v e a reduced affinity for M A K Avhicli Avas n o t obserAed.

C o n t a m i n a t i o n b y p r o t e i n s which would m a k e t h e r a d i o a c t i v e material lighter should also be ruled o u t because of t h e results o b t a i n e d w i t h a CsCl g r a d i e n t centrifuged D N A (Fig. 2c).

Difference b e t w e e n m a c r o m o l e c u l a r conformation of bulk and newly labelled D N A is t h e best h y p o t h e s i s t o explain these d a t a .

1,0

ο,β

TL.

E o 06 ■o (Ν

d

t

Λ"

2.

r V

/ ι

1 ι Γ ι

' _{ι /} / · _ι

1 / J

ι Ι ρ

Γ ο

\ / t

ψ4 ι

Ι ι

/

/

ƒ Ι ι

1;

V

12 1 β 24

FIG. 2 — Sucrose gradient centrifugation (15­5 "„) of radioactive DNA 5 min DNA : 6.4 OD units, 21,000 (3H) cts/min.

7 h DNA : 3.9 OD units. 74.000 (3H) cts/min.

5 min DNA after previous banding in CsCl density gradient : 5.1 OD units, 16,000 ('Ή) cts/min.

OD — . — . — . - : (3H) - - o - - o - -.

3.4 — Behaviour of radioactivity of 5 min and 7 h DNA in density gradients

Pneumococcal D N A eluted at alkaline p H from MAK columns Avas r e p o r t e d b y Lacks (1962) to be single-stranded. Consequently, single-stranded D N A could be expected t o be found in 5 min D N A , which gave a relatively large a m o u n t of radioactive p H fraction. Density gradients in which d e n a t u r e d D N A is shifted toAvards higher density by 0.017 g/ml in CsCl (interpolated to 42 % G-C content : Vinograd, Morris, Davidson and Dove, 1963) and by 0.026 g/ml in Cs2S04 (Erikson and Szybalski, 1964) gave clear evidence t h a t t h e

difference between neAvly labelled and bulk D N A did not involve single-stranded poly­ nucleotide chains or even denatured forms which would have been characterized b y b u o y a n t density variations. It is worth noting t h a t the one drop collection of the gradient alloAved

the detection of t h e mouse satellite b a n d in a preparatiA'e ultracentrifuge (Fig. 3). I t could be inferred from such a result t h a t a difference of about 0.003 g/cm3 in b u o y a n t density

betAveen U.V. absorbing and radioactive material would have been detected.

Moreover, a dissymetric distribution of (3H) counts compared to OD peak, which

could mean a difference in base composition of radioactive fractions, Avas not obserAed.

100 N ' f r a e t i o n *

FIG. 3 — Neutral CsCl density gradient of a five min. DNA - Total number of fractions 196 with a density gradient of about 0.001 g cm3 between two drops.

The bracket shows where denatured DNA could be expected. OD : . — . — . 3H — o — o — .

3.5 — Increased sensitivity to alkaline denaturation of radioactive material of 5 min DNA

In the course of alkaline denaturation of 7 h D N A , the ratio of (3H) counts to

U.V. absorbing material remained unchanged t h r o u g h o u t the process, pointing to a

homogeneous distribution of radioactivity among D N A molecules (Fig. 4c. d).

5 min DNA exhibited a different behaA'iour as t h e alkaline transition of the radio-actiA'ity Avas seen to occur at loAver p H t h a n t h a t of the U.V. absorbing material (Fig. 4a, /<). TAVO explanations might account for such data : slight structural modifications responsible for increased sensitivity of neAvly labelled D N A molecules to alkali titration or loAver G-C content of highly radioactiAe molecules; it has been established t h a t melting p H of d AT in 0.5 M N a+ ( I n m a n and Baldwin, 1962) is loAver t h a n melting p H of dG : dC in 0.1 M Na+

(Radding, Josse and Kornberg, 1962) (10.9 versus 11.4). Such a relationship can probably be extended to higher molarities. NeAertheless, t h e G-C content difference — if any — should be smaller t h a n 2-3 % due to our inability to find up any difference in the CsCl preparatiAc ultracentrifugation gradient betAveen OD and radioactÏAÏty p a t t e r n s (Fig. 3).

ο.α

174 iJÕ 1,66

Ο. D.

ο,β.

o,<

3H ^ 1 0 - ^ )

pH =. 11.41

**»**w

O.D O.D,

0 , 4 . .

0 , 2 . 4

1.74 1,70 1,6 6 1,6 6

FlG. 4 — Density gradient centrifugation in CsCl at alkaline p H .

Left hand diagrams (a and b) are for 5 min DNA and right hand ones (c and (/) for 7 h DNA.

O l ) 3H

3.6 — Kinetic data

Significant decrease of t h e specific actiAities of t o t a l cell D N A Avas n o t obserA'ed at six various times betAveen 5 m i n a n d 7 h after label injection and chase b y cold t h y m i ­ dine at 5 m i n u t e s (Fig. 5). These d a t a are n o t consistent Avith t h e h y p o t h e s i s of a p a r t of D N A h a v i n g a different t u r n over from b u l k D N A .

A l t h o u g h t h e specific a c t i v i t y of t o t a l D N A r a p i d l y reached a c o n s t a n t level at a b o u t 10­20 m i n u t e s after injection of t h y m i d i n e 3H , t h e r a d i o a c t i v i t y r e p a r t i t i o n in

c h r o m a t o g r a p h i c fractions Avas t i m e d e p e n d i n g over a tAvo h o u r s post injection period (Fig. 6).

At 5 m i n u t e s , t h e specific a c t i v i t y of 0.7 M fraction Avas only one third t h a t of t h e u n f r a c t i o n a t e d D N A ; it t h e n increased regularly over t h e first tAvo hours after labelling t o reach a value nearly equal t o t h a t of t o t a l D N A .

T h e 0.8 M fraction Avas r a p i d l y labelled; at 5 m i n u t e s , its specific a c t i v i t y Avas ÍAVO t o five t i m e s g r e a t e r t h a n t h a t of t o t a l D N A toAvards Avhich it t h e n decreased, r e m a i n i n g nevertheless slightly higher at 2 a n d 7 h o u r s .

T h e Ρ fraction was also r a p i d l y labelled b u t one m u s t be cautious a b o u t calculated S. A. values due t o its v e r y low c o n c e n t r a t i o n a n d t o possible release into it of c o l u m n

1 0 0

5 0 .

FIG. 5 — Specific activities of total DNA's extracted by the Schmidt and Thannhauser procedure at Aarious times after thymidine 3H injection (25 uc/mouse) and chase by 2 uM of cold thymidine 5 min after the

label injection.

For each time, ascites cells from 15 mice were pooled.

3

-H e l s / m i n / ug DNA

Cx I O "2)

6 0

r (minutes)

9 0 1 2 0 I II 4 2 0

r-FIG. 6 — Repartition of radioacti\ity in the different chromatographic fractions versus time φ 0.7 M fraction

Δ 0.8 M fraction χ pH fraction O NE part.

U.V. absorbing material. Nevertheless, one can state that its maximum S. A. was higher

than that of bulk DNA OA'er the 5-15 minutes range and then decreased.

The NE part Avas very important into 5 min DNA; nearly half of the radioactivity

could not be eluted from the column. On the other hand, into 2 and 7 h DNA's, NE part

is strongly reduced.

The oAcrall pattern of these kinetic data surprisingly pointed to the rather long

life span of newly labelled forms of DNA; one must last for more than tAvo hours after

pulse labelling to get a DNA devoid of radioactiAe material displaying a different behaviour

from total U.V. material.

4 — DISCUSSION

When DNA Avas extracted from cells AvithdraAvn after a short

in vivo

(

1I) thymidine

pulse, most of the radioactive label Avas found in a deoxyribonucleic material which differed

from bulk DNA by several criteria : sloAver sedimentation rate in sucrose gradient; higher

affinity for columns of methylated albumin; higher sensitivity to alkaline denaturation.

i.e. lowering of the pH at Avhich began the alkaline induced helix-coil transition (alkaline

CsCl density gradients). HoAveAer, at neutral pH its behaviour in Cs

SO, and CsCl density

gradients did not differ from that of bulk DNA.

As shown by the results, such a behaviour could not be accounted for only by a

difference in base composition or in molecular size of ncAvly labelled DNA, therefore it

should be related to structural modifications.

DNA intra-helical rearrangement leading to intermediati' structural states betAveen

helix and coil may be evoked; it could involve breakage or Aveakening of some hydrogen

bonds or modification of Van der Waals and liA'drophobic forces. These naturally occuring

forms may haA~e some analogy Avith intra-helical rearranged structure experimentally observed

by quite different physical techniques : microspectrophotometry (Chamberlain and Walker.

1965); spectrophotometry and viscosimetrv (Freund and Bernardi, 1963; Tikhonenko,

Perevertaylo and Dobrov. 1963) ; oscillopolarography (Palecek, 1965) ; circular dichroism

(Brahms and Mommaerts, 1964) and IOAV angle X-ray scattering (Luzzati, Mathis, Masson

and Witz. 1964). MoreoAer, the existence of DNA metastable forms related to replication

has been postulated in bacteria as Avell as in mammalian cells by Rosenberg and Cavalieri

(1964).

One can point out the remarkable stabilitA' of the replicative structures presently

described : they withstood mechanical disruption of cells and nuclei and, after extraction,

did not disappear after such treatment as ethanol precipitation, CsCl centrifugation and

proteolytic enzymes treatment. This contrasts Avith the fragility of replicative forms of

bacterial DNA (Rolfe, 1963; HanaAvalt and Ray, 1964).

An other explanation for the maintenance of the modified molecular structure can

be offered by sticking of small amounts of non DNA material related to unknown substances

or specific proteins like those demonstrated for bacteria (Jacob, Brenner and Cuzin, 1963:

Lark and Lark. 1964) Avhich seem to be involved in the physiological function of DNA.

A point of importance is to establish Avhether these replicatiA'e forms characterize

the nuclear DNA or whether they are restricted only to a part of the DNA, either

endo-genous (mitochondrial) or from symbiotic organisms.

DNA-like fibers have been visualized through electron microscope inside

mito-chondrial bodies of Ehrlich cells (Nass and Nass, 1964) and characterized into mice tissues

(Corneo. Moore, Sanadi, Grossman, Marmur, 1966). HoAvever, this deoxyribonucleic material

is quantitatively very small and can probably be neglected unless thymidine incorporation

is preferential at this cytoplasmic level; although such an eventuality is not supported

by Avell known autoradiographic data, it has recently been reported that rat liver mito­

chondrial DNA is much more rapidly labelled than nuclear DNA (Schneider and Ruff, 1965).

The replicative fractions of 5 min DNA cannot be accounted for by DNA of bacteria

or PPLO which were not, or very rarely, found in our ascites cells. The hypothesis of a

A'iral DNA cannot be completely ruled out but tAvo lines of evidence made it quite unlikely :

permanence of observations made on different lots of animals and different types of cells

(Ehrlich and Krebs ascites) Avhich could not be expected from viral cycles due to their

biological variability and host cells specificity; identical buoyant density values of radio­

active and bulk DNA Avhich implicates the same GC content for each material in a 2-3 %

range.

Finally, the best explanation for the kinetic data is that neAvly replicated DNA

is a macromolecular transient form of metabolically stable DNA into Avhich it is transformed

by structural change. It is not possible to establish Avhether such forms actually do exist

or not in the cells; their appearance from replicating DNA during cell disruption and DNA

extraction through an enzymatic equipment or any other process, remains an open

possibility.

The persistance of DNA replicative structures long after incorporation of thymidine

into polynucleotidic chains is quite an interesting fact when correlated Avith the results

obtained by Cairns (1966) through autoradiographic studies on DNA replication into

HeLa cells. This observation is in support of Cairns' conclusion that DNA synthesis speed

is loAver in animal cells than in bacterial ones. HoAveAer, if this measured speed Avas, at any

replicative point, the same and constant all

o\er

the S period, i.e. 0.5 μ/mn, our replicative

forms Avould not last more than 5 to 10 minutes. The only Avay to explain our much longer

duration is to postulate either the non constancy of speed for each replicative point or a

A ariation of speed from one point to another one depending on its location on peculiar

chromosome(s) or on some peculiar part(s) of given chromosome(s). Asynchronous speed

for DNA synthesis obserA'ed at the chromosome leAel has already been reported (Moorhead

and Defendi, 1963; Petersen, 1964; Galton and Holt, 1965).

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H . Y O S H I K A W A , and N. S U E O K A — Proc. Nat. Acad. Sci. Wash., 49 : 559 (1963).

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