N U C L E I C ACID METABOLISM I N

' H A T I N T E S T I N A L MUCOSA

by

Mary L o u i s e F l a n a g a n

B.Sc.", U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1967

A T H E S I S SUBMITTED I N P A R T I A L FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

In t h e Department

of

Biochemistry

We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e
required standard f o rthe degree.of
MASTER OF SCIENCE

The U n i v e r s i t y of British Columbia

December 1969
In p r e s e n t i n g t h i s thesis in p a r t i a l f u l f i l m e n t o f the requirements f o r

an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that

the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for r e f e r e n c e and study.

I f u r t h e r agree tha permission f o r e x t e n s i v e copying o f t h i s thesis

f o r s c h o l a r l y purposes may be granted by the Head of my Department o r

by h i s representatives. It i s understood that copying o r p u b l i c a t i o n

of this thesis f o r f i n a n c i a l gain s h a l l not be allowed without my

written permission.

Department

The U n i v e r s i t y o f B r i t i s h Columbia
Vancouver 8, Canada
 i ;

ABSTRACT .

The i n vivo synthesis of. d e o x y r i b o n u c l e i c a c i d from labelled

p r e c u r s o r s was s t u d i e d i n the r a t i n t e s t i n a l m u c o s a i n an a t t e m p t

to e l u c i d a t e t h e c o m p l e x p r o c e s s o f DNA r e p l i c a t i o n . I n one s e t
3
of experiments, t h e r a t s were i n j e c t e d with H-thymidine and t h e n

starved f o r 24 h o u r s , i n which time the stable DNA became

14
labelled with tritium. C-T:hymidine was t h e n a d m i n i s t e r e d a n d
the a n i m a l s were s a c r i f i c e d 5 minutes later. By t h i s procedure.
14
the n e w l y s y n t h e s i z e d DNA was l a b e l l e d with C.

The DNA, was f r a c t i o n a t e d by chromatography on a m e t h y l a t e d -

albumin k i e s e l g u h r column. Only one m a i n p e a k o f DNA was eluted

w i t h a sodium chloride solution r a n g i n g i n c o n c e n t r a t i o n from

0 . 5 - 0 . 6 ? .M.The t h e r m a l d e n a t u r a t i o n t e m p e r a t u r e f o r t h e DNA i n

each.fraction from t h i s p e a k was d e t e r m i n e d and t h e G + C c o n t e n t

was calculated:, W i t h i n t h e DNA p e a k o b t a i n e d f r o m MAK chromato-

graphy, the G + C'content o f t h e DNA d e c r e a s e d w i t h increasing

fraction number.

In addition t o these differences i n base composition, there

were d i f f e r e n c e s i n metabolic a c t i v i t y between t h e f r a c t i o n s ,

3 14 3 14
which were i n d i c a t e d by t h e i r H/ C ratios. The H/ C ratio

o f t h e DNA f r a c t i o n s f r o m MAK chromatography increased with

fraction number t o a maximum a t f r a c t i o n 4 o r 5 and t h e n decreased.

3

I t was f o u n d t h a t the H/O.D. r a t i o o f t h e f r a c t i o n s was n o t

constant, thus s u g g e s t i n g • t h a t the t r i t i u m might be u n e v e n l y

distributed throughout the fractions. I f the time interval

between t h e and ^ C - t h y m i d i n e injections was r e d u c e d to 3 1/2

 3 H/O.D. r a t i o became c o n s t a n t w h i l e t h e p a t t e r n o f
hours, the the

•^H/ C r a t i o s
14
remained unchanged. I f ^ C - t h y m i d i n e was administered
3 14
20 minutes before the a n i m a l s were s a c r i f i c e d , the H/ C ratio

of the DNA f r a c t i o n s f r o m MAK chromatography i n c r e a s e d with

increasing fraction number. From t h e s e r e s u l t s i t was concluded

that' s m a l l m o l e c u l a r weight, newly s y n t h e s i z e d DNA, which was

highly labelled with 1 4

C, was being incorporated with time into

the high molecular weight, s t a b l e DNA f r a c t i o n , which i s labelled

with 3
H.
During these experiments i t was observed that the pattern
3 14 • .
of H/ C ratio versus fraction number v a r i e d a c c o r d i n g to the

treatment given t o t h e DNA sample p r i o r to the'preparation for

radioactive counting. I f the s a m p l e was d e n a t u r e d by heating

to obtain its v a l u e , and then/dialyzed". a g a i n s t distifled '

water, s m a l l m o l e c u l a r weight nucleotides passed into the

dialysate.

The d e n a t u r e d DNA sample a l s o gave d i f f e r e n t r e s u l t s from

the n a t i v e DNA s a m p l e on digestion with s n a k e venom p h o s p h o d i e s -

terase. On' the denatured sample, the pattern of r e l e a s e of 3

H
14 4

and C labelled material i n t o the acid soluble material,

indicated that both these l a b e l s were u n i f o r m l y distributed

along the DNA chain. On t h e o t h e r h a n d , w i t h t h e n a t i v e 5 m i n .

•' 14
DNA s a m p l e s , the r e l e a s e of C labelled material i n t o the acid

s o l u b l e f r a c t i o n was that expected f o r DNA w h i c h had incorporated

^ C - p r e f e r e x i t i a l l y i n t o the 3* t e r m i n a l p o s i t i o n s .
The s e p a r a t i o n ' o f t h e p y r i m i d i n e c l u s t e r s o f DNA indicated
' 14 3
that t h o s e were n o t u n i f o r m l y labelled with C and H.
 iii

"ACKNOWLEDGEMENTS

The author wishes t o express her sincere thanks and

appreciation t o D r . S. H. Z b a r s k y for his continual a d v i c e and

encouragement.

It i s a p l e a s u r e t o acknowledge t h e p e r s o n a l a s s i s t a n c e

of t h e N a t i o n a l Research C o u n c i l and t h e M e d i c a l Research

Council i n t h e form o f S t u d e n t s h i p s .

cO
 iv

TABLE OF CONTENTS
Page

INTRODUCTION 1

E a r l y Experiments 1

DNA R e p l i c a t i o n 5

Terminal Incorporation of Nucleotides 8

I s o l a t i o n o f DNA 12

The P h y s i c a l and C h e m i c a l Heterogeneity o f DNA ...... 15

Metabolic Heterogeneity • 19

The Present Investigation 23,

MATERIALS AND METHODS . 26

Administration of Labelled Material 26

I s o l a t i o n o f DNA from R a t I n t e s t i n a l Mucosa 26

C h r o m a t o g r a p h y o f t h e DNA solution on Methylated

Albumin K i e s e l g u h r . .. 28 •

Thermal D e n a t u r a t i o n Curves 30

Radioactive Counting Procedures 31

EXPERIMENTAL 36
. ' <P

Administration of L a b e l l e d Thymidine P r e c u r s o r s ..... 36

Characterization of the Isolated DNA 37

Chromatography o f t h e I s o l a t e d DNA o n

Methylated-Albumin Kieselguhr 38

Thermal D e n a t u r a t i o n Curves 39

R a d i o a c t i v e C o u n t i n g o f D o u b l y - L a b e l l e d DNA 44
D e g r a d a t i o n o f t h e I s o l a t e d DNA.by Snake Venom
Phosphodiesterase 64
The P u r i f i c a t i o n and C o u n t i n g o f t h e I n t e r p h a s e
Layers o f the Tissue Extracts 70
 V

• Page

I s o l a t i o n and C h a r a c t e r i z a t i o n of the
Pyrimidine Isostichs •••• 70

SUMMARY • ••• 7 9

BIBLIOGRAPHY «... 83
 vi

TABLES
Page

I. T v a l u e s and G-C c o n t e n t o f s e p a r a t e fractions

o r t h e m a i n peak a f t e r c h r o m a t o g r a p h y of
DNA f r o m i n t e s t i n a l mucosa 44

II. The r a t i o o f r a d i o a c t i v i t y due t o " ^ H / O p t i c a l

D e n s i t y o f the DNA f r a c t i o n s o b t a i n e d from^
MAK C h r o m a t o g r a p h y i n e x p e r i m e n t s i n w h i c h H-
t h y m i d i n e was a d m i n i s t e r e d t o r a t s 24 h o u r s b e f o r e ^6
the i n j e c t i o n o f C-thymidine

III. E f f e c t o f v a r i o u s t r e a t m e n t s on t h e r a d i o a c t i v i t y
and a b s o r b a n c e o f a s o l u t i o n o f d o u b l y - l a b e l l e d
r a t i n t e s t i n e DNA i n s a l i n e c i t r a t e 57

IV. Radioactivity and a b s o r b a n c y a f t e r d i a l y s i s

a g a i n s t d i s t i l l e d water o f f r a c t i o n s o f
d o u b l y - l a b e l l e d D N A o b t a i n e d by chromatography
on M A K . . . 58

V. Radioactivity and a b s o r b a n c y a f t e r h e a t i n g and

then d i a l y s i s a g a i n s t d i s t i l l e d water o f •
f r a c t i o n s o f d o u b l e — l a b e l l e d rDNA o b t a i n e d by
c h r o m a t o g r a p h y on M A K 59

VI. Radioactivity and a b s o r b a n c y o f t h e p y r i m i d i n e

i s o s t i c h s i s o l a t e d by c h r o m a t o g r a p h y on
. DEAE-Cellulose of the formic acid-diphenylamine
h y d r o l y s a t e o f m u c o s a l D N A , w h i c h was a l l o w e d
to.incorporate H f o r 3 1 / 2 h o u r s and C for
5 minutes .. 74-

VII. Radioactivity and a b s o r b a n c y o f t h e p y r i m i d i n e

i s o s t i c h s i s o l a t e d by c h r o m a t o g r a p h y on
DEAE-Cellulose of the formic a c i d - diphenylamine
h y d r o l y s a t e o f m u c o s a l D N A w h i c h was a l l o w e d
to incorporate H f o r 3 1 / 2 h o u r s and C for
10 m i n u t e s . • . ?7
 vii

FIGURES

Page

1. '.L--: KornBerg*s .hodel of DNA.' replication ...•.».<•<• ?•• 9

2. Suggested mechanism f o r t e r m i n a l incorporation

o f n u c l e o t i d e s I n t o DNA 11

3. Integral discriminator bias curves f o r

. s t a n d a r d H ( H" 0) and
2 C (Na„ C 0 ) obtained
3

from the r e d s c a l e r o f the Packard T r i Carb

314 AX L i q u i d S c i n t i l l a t o r S p e c t r o p h o t o m e t e r

4. Integral discriminator bias curves for

standard H and C d e t e r m i n e d on t h e g r e e n
s c a l e r o f t h e P a c k a r d T r i C a r b 3 1 4 AX
L i q u i d S c i n t i l l a t o r Spectrophotometer.
The c u r v e s r e p r e s e n t t h e a v e r a g e o f t h e
d e t e r m i n a t i o n o f s i x d i f f e r e n t window s e t t i n g s . ..

5. C h r o m a t o g r a p h y o f DNA f r o m r a t i n t e s t i n a l
- mucosa on a MAK c o l u m n . E l u t i o n was c a r r i e d
out w i t h a g r a d i e n t o f N a C l i n 0.05 M p h o s p h a t e
b u f f e r , pH 6 . 7 40

6. E f f e c t o f i n c r e a s i n g t e m p e r a t u r e on t h e o p t i c a l
density of doubly-labelled r a t i n t e s t i n a l
m u c o s a l DNA. 43
3 14
7. Ratio of H cpm/ C cpm i n f r a c t i o n s c o m p r i s i n g
t h e DNA p e a k e l u t e d by c h r o m a t o g r a p h y o f d o u b l y -
l a b e l l e d m u c o s a l DNA, w h i c h h a d b e e n a l l o w e d t o
incorporate H f o r 24 h o u r s , on MAK c o l u m n s 45
3
8. C h r o m a t o g r a p h y o f m u c o s a l DNA, J a b e l l e d w i t h H
- t h y m i d i n e f o r 3 1 / 2 h o u r s and C-thymidine
f o r 5 m i n u t e s , on an MAK c o l u m n ... 48
9. Chromatography o f mucosal D N A , ^ a b e l l e d wi t h 3
H-
t h y m i d i n e f o r 3 1 / 2 h o u r s and C-thymidine
for 10 m i n u t e s , on an MAK c o l u m n 4-9
3
•10. The r a t i o o f r a d i o a c t i v i t y due t o H/optical
d e n s i t y o f t h e DNA f r a c t i o n s o b t a i n e d f r o m
MAK c h r o m a t o g r a p h y i n e x p e r i m e n t s i n w h i c h
H - t h y m i d i n e was a d m i n i s t e r e d t o r a t s 3 1 / 2 50
h o u r s b e f o r e i n j e c t i o n o f """^C-thymidine.

11. The ratio of H/ C i n f r a c t i o n s c o m p r i s i n g the

DNA p e a k e l u t e d b y ^ c h r o m a t o g r a p h y o f DNA w h i c h
was labelled with H-thymidine f o r 3 1/2 hours.
 Vlll

Page

3 14
12. The r a t i o o f H/ C i n f r a c t i o n s comprising the
DNA peak e l u t e d b y ^ c h r o m a t o g r a p h y - o f DIjIA w h i c h
was l a b e l l e d w i t h H f o r 24 h o u r s and C for
20 m i n u t e s . The c o m p a r i s o n i s made between ;p
<;

f r a c t i o n s t h a t a r e d e n a t u r e d by h e a t i n g ( c h r o m a t o -
g r a p h y I b e f o r e r a d i a t i o n c o u n t i n g and t h o s e w h i c h
h a d no t r e a t m e n t p r i o r t o c o u n t i n g 55

13. C h r o m a t o g r a p h y on D E A E - C e l l u l o s e o f t h e
d i s t i l l e d w a t e r d i a l y s a t e o f t h e m u c o s a l DNA
t h a t h a d b e e n d e n a t u r e d by h e a t i n g p r i o r t o
dialysis. E l u t i o n was c a r r i e d o u t w i t h a
g r a d i e n t o f N a C l i n 7M u r e a - T r i s HC1.
pH 7. 8 t . 62

14. Release of u l t r a v i o l e t absorbing material

and r a d i o a c t i v i t y by d i g e s t i o n o f d o u b l y -
l a b e l l e d m u c o s a l DNA w i t h s n a k e venom
phosphodiesterase 67 (a)

1'5. C h r o m a t o g r a p h y on D E A E - c e l l u l o s e o f the
diphenylamine-formic a c i d hydrolysate o f
DNA, w h i c h had b ^ | n l a b e l l e d w i t h H for
3 1/2 h o u r s and C f o r 5 minutes.
E l u t i o n was c a r r i e d o u t w i t h a g r a d i e n t o f
L i C l i n l i t h u i m a c e t a t e - pH .5.3 75

16. C h r o m a t o g r a p h y on D E A E - c e l l u l o s e o f t h e
diphenylamine-formic acid hydrolysate-
pf DNA, w h i c h h a d begn l a b e l l e d w i t h H
for 3 1/2 h o u r s and C f o r 10 m i n u t e s .
E l u t i o n was c a r r i e d o u t w i t h a g r a d i e n t
of L i C l i n l i t h u i m a c e t a t e - pH 5.3 . 76
 ix

• L I S T OF ABBREVIATIONS

DNA deoxyribonucleic acid

5 min.DNA DNA w h i c h was a l l o w e d to.incorporate ^ C~

4

thymidine f o r 5 min.

10 m i n . DNA DNA w h i c h was a l l o w e d t o i n c o r p o r a t e ^" C~4

t h y m i d i n e f o r 10 m i n .

20 m i n , DNA DNA w h i c h was a l l o w e d t o i n c o r p o r a t e "*"C- 4

t h y m i d i n e f o r 20 m i n .

A adenine

G guanine

C cytosine

T thymine

MAK a c h r o m a t o g r a p h i c column of methylated-

albumin kieselguhr.

mCi millicurie

umole. micromole

nm nanometer

cpm " c o u n t s per. m i n u t e

O.D. optical density

p p
O 2\6^d'iphehyl oxazole _-

-dimethyl POPOP l,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene

CME-carbodiimide N-cyclohexyl-'N-[4-methylmorpholinum)
•ethylcarbodiimide
 1

INTRODUCTION

The l a s t twenty y e a r s have seen f u l l r e a l i z a t i o n o f the

great b i o l o g i c a l importance o f the n u c l e i c acids^, f i r s t i s o l a t e d

by Miescher (1) i n t h e 1870's and i n c r e a s i n g knowledge o f the

chemical and p h y s i c a l p r o p e r t i e s o f d e o x y r i b o n u c l e i c a c i d has

strengthened the e a r l y b e l i e f t h a t DNA was the t r a n s m i t t e r o f

genetic information. The u n r a v e l l i n g o f the complex process o f

h e r e d i t y has been made p o s s i b l e as a r e s u l t o f the i d e n t i f i c a t i o n

of the s t r u c t u r e and f u n c t i o n s o f DNA.

The s t r u c t u r e s o f the n u c l e o t i d e components had been

determined by chemical means (2) when Watson and Crick,

postulated a double h e l i c a l s t r u c t u r e f o r DNA i n 1952 ( 3 ) .

From t h i s model, a s e m i - c o n s e r v a t i v e mechanism o f r e p l i c a t i o n

was proposed i n which each s t r a n d o f the double h e l i x a c t s as

a template f o r the formation o f a new s t r a n d . While this

developed, f u r t h e r i n f o r m a t i o n was a v a i l a b l e which was p o i n t i n g

t o the r o l e o f DNA i n h e r e d i t y , which had been suspected

because o f the m e t a b o l i c s t a b i l i t y and c e l l u l a r l o c a t i o n o f t h i s

nucleic acid. Three types o f experiments confirmed.this

f u n c t i o n o f DNA i n h e r e d i t y . First/ the h i g h e s t r a t e o f

mutations i n b a c t e r i a induced by u l t r a v i o l e t l i g h t o c c u r r e d a t

wave lengths c o r r e s p o n d i n g t o the a b s o r p t i o n maxima o f n u c l e i c

acids (4). Second/ Avery e t a l . (5) e x t r a c t e d DNA from a

capsule forming s t r a i n Cs) o f pneumococcus, added i t t o a

c u l t u r e o f a non-capsule forming s t r a i n (R) and found t h a t the

l a t t e r had a c q u i r e d the a b i l i t y t o form c a p s u l e s . Third,

 2

H e r s h e y and. C h a s e ( 6 ) i n f e c t e d b a c t e r i a w i t h phage, the

35 32
protein o f w h i c h was l a b e l l e d with S and the DNA with P,

and studied the d i s t r i b u t i o n of the isotope in bacteria. They

concluded that only the DNA of the phage e n t e r e d the b a c t e r i a l

cell.

E x p e r i m e n t s - i n DNA m e t a b o l i s m were done as early as

1940 by von Euler and von Hevesy (7), who measured the uptake
32
of ">P i n t o DNA
in vivo. T h e r e was a p o s i t i v e c o r r e l a t i o n
32
between the uptake o f P by a t i s s u e and i t s m i t o t i c a c t i v i t y ,
32
suggesting t h a t the uptake of P o c c u r r e d as a r e s u l t o f
• 32

synthesis of DNA which accompanied m i t o s i s . The values of P

uptake obtained, however, i n d i c a t e d t h a t t w i c e , as much DNA v/as

b e i n g . - p r o d u c e d as w o u l d be expected from the increase in tissue

mass, and therefore DNA was considered t o have a continuous

turnover. This contradicted the f i n d i n g of Brues (8) that DNA

i s -a v e r y stable cell constituent. As the.total activity of the

32 32

P i n DNA was constant, the large P u p t a k e was thought to be

due t o exchanges i n the terminal nucleotides as w e l l as to net

synthesis.
Interest i n the b i o s y n t h e t i c mechanism can be traced back
t o the e x p e r i m e n t s o f B a r n e s and Schoenheimer (9). They found
15 '

that N i n ammonium c i t r a t e appeared i n nucleic acids and that

urea,, h i s t i d i n e and arg i i i n e are not d i r e c t precursors in

purine synthesis. Since then, the complete b i o s y n t h e t i c scheme

has been w o r k e d o u t for purines by B u c h a n a n and colleagues (10)

and for pyrimidines by Reichard and co-workers (10). This has

 a l s o been f o l l o w e d by s t u d i e s on the i n c o r p o r a t i o n of nucleosides

and n u c l e o t i d e s i n t o p o l y n u c l e o t i d e s t r u c t u r e s . In these

i n v e s t i g a t i o n s , thymidine has been s t u d i e d e x t e n s i v e l y because

i t occurs i n DNA and not t o any great extent i n RNA.

Brown (11) d e t e c t e d a s m a l l i n c o r p o r a t i o n of thymine

1, 3 - i n t o the p o l y n u c l e o t i d e thymine component of

regenerating rat liver. T h i s r e s u l t was a l s o o b t a i n e d by Adams'

(12). On the o t h e r hand, P l e n t l and Schoenheimer (13) along

w i t h 'other workers found thymine to be i n e f f e c t i v e as a n u c l e i c

acid precursor. JiThyihine.- i s degraded to R-amino i s o b u t y r i c

a c i d which i s n o t i n c o r p o r a t e d i n t o DNA (10)..

U n l i k e thymine, the n u c l e o s i d e thymidine can be utilized

because thymine i s degraded b e f o r e i t can be used and t i s s u e s

have the n e c e s s a r y enzymes f o r phosphoryla.ting thymidine to

thymidine t r i p h o s p h a t e which i s the p r e c u r s o r f o r DNA. (10)/

The Enzymatic S y n t h e s i s of DNA

A f t e r the n u c l e i c a c i d p r e c u r s o r s were i d e n t i f i e d by

r a d i o a c t i v e t r a c e r s , the next s t e p i n the e l u c i d a t i o n o f DNA

s y n t h e s i s was t h e i s o l a t i o n of the enzymes i n v o l v e d . Using

P u r i f i e d enzymes, the r e a c t i o n was determined (15) as: a)

the. b i o s y n t h e s i s o f p u r i n e and p y r i m i d i n e r i b o n u c l e o s i d e

monophosphates b) the p h o s p h o r y l a t i o n of these monophosphates

t o the diphosphate stage and the c o n v e r s i o n o f these r i b o n u c l e o -

t i d e s t o the c o r r e s p o n d i n g deoxyribonucleotides. c) the

p h o s p h o r y l a t i o n o f the d e o x y r i b o n u c l e o s i d e diphosphates to the

 4

triphosphate stage and d) the polymerization of the

deoxyribonucleoside triphosphates to give deoxyribopolynucleo-

tides. This l a s t polymerization can only take place i n the

presence of an appropriate DNA primer.

The complex processes involved i n t h i s l a s t step have

been c l a r i f i e d by the i s o l a t i o n of DNA polymerase from E_. c o l i

by Kornberg (15). He noted that, i n the presence of the four

deoxyribonucleoside triphosphates, a p u r i f i e d enzyme preparation

catalyzes the r e p l i c a t i o n of a DNA primer. The following

reaction summarizes t h i s process:

n dTP'PP dTP
m dG PPP
+
Mg ++
dGP
n dA PPP
+ + D N A
P r i m e r
> D N A
- dAP + 2 (m+n)

m dC PPP polymerase dCP m+n

The r a t i o of the amount of adenine plus thymine to

A +T
guanine plus cytosine (^ + ^) i n the newly synthesized DNA i s

equal t o that of the primer. Varying the r e l a t i v e q u a n t i t i e s

of the four precursors does not influence the composition of

the newly formed DNA (16). Unnatural bases can be incorporated

i n t o the new DNA e.g. 5-hydroxymethyl cytosine f o r cytosine,

u r a c i l or 5-bromouracil f o r thymine and hypoxanthine f o r

guanine, provided these are i n the system as deoxyribose

triphosphates in.the presence of the other nucleoside t r i p h o -

sphates. Therefore, the DNA polymerase catalyzes the b i o -

s;yrthesis of DNA, but the s p e c i f i c i t y , with regards to the

base sequence o f the newly formed DNA molecule, i s determined

 5

by the DNA primer. S p e c i f i c k i n a s e s are p r e s e n t which

phosphorylate o n l y the c o r r e c t bases to the deoxyriboside

t r i p h o s p h a t e l e v e l and they determine' t h a t the newly-formed

DNA always has the i d e n t i c a l base sequence as the primer (16).

The DNA polymerase can r e c o g n i z e the p o s i t i o n and s t r u c t u r e of

t h e pentose phosphate m o i e t i e s of the deoxyribonucleoside

t r i p h o s p h a t e s , but not the v a r i o u s bases.

^ I f n a t i v e h e l i c a l DNA i s used as a template with the

p u r i f i e d enzyme, the product i s s i m i l a r t o n a t i v e DNA except

f o r i t s unusual c a p a c i t y to resume a h e l i c a l conformation after

denaturation (16). The product a l s o has a branched s t r u c t u r e

when examined w i t h an e l e c t r o n microscope. New s t r a n d s are not

C o v a l e n t l y bound to the template and can i n t e r n a l l y hydrogen-

bond t o form a h a i r p i n or p l e a t e d s t r u c t u r e (16).

DNA R e p l i c a t i o n

B a s i c a l l y t h e r e are t h r e e p o s s i b l e methods o f r e p l i c a t i o n :

a) c o n s e r v a t i v e , i n which the parent molecule remains i n t a c t

and one e n t i r e newly-formed daughter molecule i s formed, b)

s e m i - c o n s e r v a t i v e , i n which one s t r a n d o f each c h a i n o r i g i n a t e s

f r o m t h e parent molecule and the o t h e r i s newly s y n t h e s i z e d ,

c) d i s p e r s i v e i n which p a r e n t a l and o f f s p r i n g components a r i s e

f r o m p a r t s o f a l l DNA molecules.

The s e m i - c o n s e r v a t i v e method was suggested by Watson and

Crick (3). R e p l i c a t i o n c o u l d be achieved by the unwinding of

t h e two s t r a n d s w i t h c o n c u r r e n t attachment o f the complementary

 6

deoxynucleoside triphosphates to each parental strand. After

one cycle of replication, there w o u l d be two DNA molecules of

identical composition and sequence, each containing one strand

of t h e original DNA.

The classical experiment of Meselson and Stahl (17)

Supported t h i s theory. B a c t e r i a , whose DNA was labelled with

^N, were allowed to replicate twice in a culture medium

containing ^ N.
4
After each cycle of replication, the DNA

isolated from these b a c t e r i a was p l a c e d in a s o l u t i o n of CsCl

15
and centrifuged until e q u i l i b r i u m was reached. The • N -
labelled DNA travelled further down t h e centrifuge tube than
14 14

the N - labelled DNA o r h y b r i d DNA which contained both N

and ^N. Thus t h e three types of DNA c o u l d be separated and

identified because of d i f f e r e n c e s in their buoyant densities.

From t h e d a t a o b t a i n e d f r o m t h e d e n s i t y g r a d i e n t equilibration,

they c a l c u l a t e d t h a t after two c y c l e s of replication, half the

15 14
DNA molecules contained 50% N a n d 50% N, while the other
14
half' c o n t a i n e d o n l y N. These results would be predicted for

a semi-conservative method of replication.

There i s a d i f f i c u l t y i n a p p l y i n g t h i s theory to the

circular DNA found i n bacteria, viruses, a n d r a t ' . l i v e r - • m i t o c h o n d r i a (IE

Cairns (19) a p p r o a c h e d t h e p r o b l e m b y u s i n g autoradiography

techniques and e l e c t r o n microscopy. Bacteria were allowed to

replicate in a medium c o n t a i n i n g 3
H - thymidine i n contact with

photoradiographic emulsion. The 0 particles emitted from the

tritium reacted to produce dark spots i n the emulsion. The

electron micrographs of this process indicated that replication

 7

o f the molecule s t a r t e d a t one p o i n t i n the c i r c l e . The

formation o f 2 new DNA s t r a n d s r e s u l t e d i n a f o r k , w i t h the ends

o f the f o r k b e i n g joined together. R e p l i c a t i o n proceeded

around the molecule u n t i l two daughter DNA m o l e c u l e s had been

formed.

Dounce (20) c a l c u l a t e d the r a t e a t which the DNA molecule

must unwind i n o r d e r f o r the e n t i r e molecule t o r e p l i c a t e

w i t h i n the time i n t e r v a l o f DNA r e p l i c a t i o n . T h i s r a t e seemed

e n e r g e t i c a l l y unfavourable and he t h e r e f o r e proposed an

a l t e r n a t e mechanism. He suggested t h a t o n l y one s t r a n d o f the

molecule i s c o p i e d , the other s t r a n d being prevented from doing

so by a h i s t o n e , a b a s i c p r o t e i n which i s known t o b i n d t o DNA

i n the n u c l e u s . Butler (21) had e a r l i e r proposed a mechanism

t o account f o r the unwinding o f the DNA h e l i x . He p i c t u r e d the

DNA polymerase as a d i s k w i t h two s l o t s , one f o r each o f the

DNA primer s t r a n d s . The d i s k would a t t a c h i t s e l f t o a l o o s e

end o f t h e molecule w i t h the end o f one s t r a n d i n each h o l e .

As t h e complementary n u c l e o t i d e a t t a c h e s t o t h e growing c h a i n ,

the d i s k moves along the DNA h e l i x , r o t a t i n g as i t p r o g r e s s e s ,

c a u s i n g the double h e l i x t o unwind.

I n c o n t r a s t t o these t h e o r i e s , C a v a l i e r i and Rosenberg

(22) proposed a mechanism/ which i s not w i d e l y h e l d , i n which

t h e r e i s no s t r a n d separation.

Kornberg and h i s c o l l e a g u e s (23) r e c e n t l y have suggested

a model f o r DNA polymerase by which i t i s e x p l a i n e d how the

enzyme can achieve r e p l i c a t i o n o f both s t r a n d s even though the

 8

enzyme operates o n l y i n the 5 ' > 3 1

direction. In

a d d i t i o n to i t s r e p l i c a t i v e a c t i v i t y , the DNA polymerase i s

b e l i e v e d to have a degree of h y d r o l y t i c a c t i v i t y . Thus the

DNA polymerase c o u l d c a t a l y z e the i n t r o d u c t i o n of a n i c k i n

the DNA c h a i n , which would i n i t i a t e r e p l i c a t i o n . Replication

would then proceed by c o v a l e n t e x t e n s i o n of the 3'OH end. The

5 ' end may be degraded to some e x t e n t by a 5 1

>3' nuclease

a c t i o n or may a t t a c h t o some membrane s i t e . After replication

has proceeded f o r a c e r t a i n d i s t a n c e , i t switches to the

complementary s t r a n d as a template to form a fork., which i s then

c l e a v e d by an endonuclease. A r e p e t i t i o n o f t h i s sequence l e a d s

to i n t e r r u p t i o n s or s m a l l p i e c e s of DNA near the . r e p l i c a t i n g

fork. L i g a s e c o u l d then s e a l these interruptions. Rather

than a simultaneous s e q u e n t i a l r e p l i c a t i o n o f b o t h s t r a n d s , this

scheme i m p l i e s a r e p l i c a t i o n which i s s t a g g e r e d , a l t e r n a t i n g

from one s t r a n d t o the other as i l l u s t r a t e d i n Figure 1.

.Terminal I n c o r p o r a t i o n of Nucleotides

In e a r l y s t u d i e s of DNA polymerase a c t i o n , A d l e r e t a l .

(24).noted t h a t i f one of the d e o x y r i b o n i i c l e o t i d e s was lacking,

t h e r e was o n l y l i m i t e d i n c o r p o r a t i o n of the r e m a i n i n g nucleotides

i n t o the primer. Degradation by snake venom phosphodiesterase;

of the product formed under these c o n d i t i o n s suggested t h a t

t h e r e was a l i m i t e d i n c o r p o r a t i o n of one or two nucleotides

i n t o the 3'OH end.

Krakow e t a l . (25) i s o l a t e d an enzyme from c a l f thymus

n u c l e i which c a t a l y z e s the t e r m i n a l i n c o r p o r a t i o n of n u c l e o t i d e s
 9.

Nicked Covalent Formation

reckon extension oP Pork

& nuclease
cleavage

F i g . 1. The mechanism of a c t i o n of DNA polymerase i n DNA r.epli6atIon

according t o Korriberg (23).
 10

i n t o the p r i m e r . T h i s enzyme has been c a l l e d t e r m i n a l nucleo-

t i d y l t r a n s f e r a s e and r e q u i r e s heat denatured DNA as a primer,

Mg i o n s and c y s t e i n e . Keir (26) suggested t h a t the t e r m i n a l

enzyme i s a c a t a l y t i c subunit o f the DNA polymerase because

of f i n d i n g s t h a t the t e r m i n a l enzyme has a lower molecular

weight and t h a t there are two o p t i m a l Mg + +

concentrations f o r

t e r m i n a l a d d i t i o n c a t a l y z e d by a p r e p a r a t i o n which contains

both r e p l i c a t i v e and t e r m i n a l n u c l e o t i d y l t r a n s f e r a s e

activities. I t was suggested (26) t h a t the parent enzyme

d i f f e r e d from the s u b u n i t s i n terms o f c o n f i g u r a t i o n and

d i s p o s i t i o n o f c o f a c t o r s and s u b s t r a t e s a t the a c t i v e s i t e .

32

Studies o f the d i s t r i b u t i o n o f P i n nucleotides

.following incubation with the l a b e l l e d t r i p h o s p h a t e s suggested

t o A d l e r e t a l . (24) t h a t hydrogen bonding i s necessary f o r

t e r m i n a l i n c o r p o r a t i o n i f the two s t r a n d s are o f d i f f e r e n t lengths

As I l l u s t r a t e d i n Figure 2, the f i r s t n u c l e o t i d e i s c o v a l e n t l y

bonded t o the 3'OH end o f the s h o r t e r c h a i n a t a p o i n t which

permits i t t o be hydrogen bonded t o i t s a p p r o p r i a t e nucleo-

t i d e i n the o p p o s i t e strand according t o the complementary base

pairing principle. Only one n u c l e o t i d e i s added i f the base

i n the p a i r i n g p o s i t i o n w i l l not hydrogen bond w i t h the base i n

the o p p o s i t e strand. The f u n c t i o n o f t h i s l i m i t e d incorporation

is.unknown, but i t may be t o r e p a i r the s h o r t e r s t r a n d o f a

double h e l i x i n which the two s t r a n d s are o f unequal lengths

(24) . '
nucleotides into PNA.
Isolation of DNA

DNA i s l o c a t e d mainly, i n the nucleus of cells., where i t

i s bound t o p r o t e i n . I n 1868-69, M i e s c h e r (1) first isolated

nucleic a c i d s by digesting pus cells with pepsin-hydrochloric

acid and then shaking the d i g e s t e d mixture with ether to give

a nuclear fraction, w h i c h c o u l d be filtered off. From the

n u c l e a r m a t e r i a l , he isolated nucleic a c i d s which are s o l u b l e

in d i l u t e alkali but insoluble in dilute acid. In present day

methods, p r o t e i n i s r e m o v e d by detergents, isoamyl alcohol/

chloroform or phenol. R i b o s o m a l RNA i s precipitated by a

solution o f 1.0, bl i o n i c strength. The method o f C o l t e r e t al_.

(27), which i s f o l l o w e d i n t h i s investigation, utilizes sodium

deoxycholate as a detergent t o d i s r u p t membrane s t r u c t u r e s and

phenol t o denature p r o t e i n , most i m p o r t a n t of which i s

deoxyribonuclease. The isolated DNA can be purified and

characterized by a variety of chromatographic techniques.

One of the earliest methods u s e d to separate different

types of DNA utilized the solubility o f v a r i o u s DNA species i n

Physiological saline a t 85°C (28) or i n a l k a l i (29). Some

s e p a r a t i o n was a l s o o b t a i n e d by differential centrifugation (30)

and c h r o m a t o g r a p h y on h i s t o n e columns (31). Cesium chloride

gradient centrifugation has been used mainly as an a n a l y t i c a l :'

procedure, since i t allows s e p a r a t i o n o f DNA molecules with

different d e n s i t i e s (17).

Fractionation o f DNA by counter current distribution

has had a limited use. I n 1956, using this procedure, Albertsson

(32) separated single stranded from double stranded DNA with

 13

an e q u i r a o l a r N a H P 0
2 4 and N a H P 0
2 4 phosphate s o l u t i o n pH6.8-7.0

c o n t a i n i n g 4.4% p o l y e t h y l e n e g l y c o l and 7.0% d e x t r a n . With a

Solution o f 0.7% sodium d e x t r a n s u l f a t e and 2% m e t h y l c e l l u l o s e '

i n O.B'M N a C l o r pH 7.0, F r i c k and L i f (33) o b s e r v e d that the

p a r t i t i o n c o e f f i c i e n t between t h e phases o f a sodium d e x t r a n

s u l f a t e - m e t h y l c e l l u l o s e aqueous system v a r i e d w i t h the

molecular size of the nucleic acid. The n a t u r e o f t h e s e p a r a t i o n •

i s n o t c l e a r , b u t DNA c o n t a i n i n g ,a h i g h p e r c e n t a g e o f adenine

and thymine %s -'ca-rri-e'd in--' thev o r g a n i c pliase.

'Bendich- e t • al.".'- (: 4 8.?/ separated the transforming

a c t i v i t i e s o f pneumococcal DNA on columns o f ECTEOLA c e l l u l o s e .

The t r a n s f o r m i n g a c t i v i t i e s were n o t r e s t r i c t e d t o any one

c h r o m a t o g r a p h i c peak o r r e g i o n , b u t were p r e s e n t i n s e v e r a l o f

the DNA f r a c t i o n s , w h i c h had been o b t a i n e d by e l u t i o n w i t h

solutions o f w i d e l y v a r y i n g i o n i c s t r e n g t h and pH. Rechromato-

graphy d i d n o t . g i v e r e p r o d u c i b l e r e s u l t s .

F r a c t i o n a t i o n o f DNA on h y d r o x y a p a t i t e columns has n o t

been v e r y s u c c e s s f u l . However, by v a r y i n g t h e c o n d i t i o n s o f
•

f r a c t i o n a t i o n , McCallum r e p o r t e d (35) c o r r e s p o n d i n g d i f f e r e n c e s

in fractionation. S e p a r a t i o n a c c o r d i n g t o base composition

resulted f r o m t h e " t h e r m a l " chromatography o f n a t i v e DNA w i t h

an e l u t i n g . s o l u t i o n o f c o n s t a n t phosphate c o n c e n t r a t i o n .

R e n a t u r e d DNA c o u l d be i s o l a t e d f r o m d e n a t u r e d DNA by chromato-

graphy a t h i g h t e m p e r a t u r e s and u s i n g a s a l t g r a d i e n t i n t h e

eluting solution. A t low t e m p e r a t u r e s , denatured DNA i s

separated f r o m n a t i v e o r r e n a t u r e d DNA due t o i t s a b i l i t y t o

 14

form i n t r a m o l e c u l a r secondary s t r u c t u r e s . Bernardi (36) was not

a b l e to f u r t h e r f r a c t i o n a t e n a t i v e DNA on hydroxyapatite

Columns a c c o r d i n g t o d i f f e r e n c e s i n base composition or b i o -

logical properties. Some f r a c t i o n a t i o n was achieved on the

b a s i s o f molecular size.

The b i n d i n g of DNA to n i t r o c e l l u l o s e membrane f i l t e r s

has been used p a r t i c u l a r l y f o r the i s o l a t i o n of DNA-RNA h y b r i d s .

Oberg e t a l . (37) have r e p o r t e d the s e p a r a t i o n o f double stranded

f r o m s i n g l e stranded DNA by g e l f i l t r a t i o n on pearl-condensed

agarose. A s i m i l a r s e p a r a t i o n f o r l a r g e q u a n t i t i e s of m a t e r i a l

can be o b t a i n e d on columns o f s i l k fibroin (38). The

r e v e r s i b l e b i n d i n g o f DNA to polymethacrylate has been used to

f r a c t i o n a t e DNA a c c o r d i n g to base composition (39).

DEAE-Cellulose i s used e x t e n s i v e l y f o r s e p a r a t i n g

o l i g o n u c l e o t i d e s r a t h e r than h i g h e r m o l e c u l a r weight DNA

molecules. More r e c e n t l y , Cerny e t a l . (40) separated pyrimidine

isostichs on a D E A E - c e l l u l o s e column a c c o r d i n g to c h a i n length

a t pH 5.3 and a c c o r d i n g to base composition on another column

a t pH 3.1.

The most w i d e l y used column f o r s e p a r a t i n g n u c l e i c a c i d s

i s t h e methylated albumin - K i e s e l g u h r (M.A.K.) column. The

p r e p a r a t i o n and use of t h i s m a t e r i a l was f i r s t described i n 1955

by Lerman (41). By m o d i f y i n g the m a t e r i a l s and p r e p a r a t i o n o f

the column, M a n d e l l and Hershey (42) were a b l e to f r a c t i o n a t e

n u c l e i c a c i d s a c c o r d i n g to t h e i r m o l e c u l a r size. T_ DNA was

shown t o c o n s i s t o f one or more l a r g e m o l e c u l e s o f identical

 15

l e n g t h which c o u l d be e x t r a c t e d by p h e n o l w i t h o u t breakage.

Other workers (43,44) h a d reported that T 2 DNA was a mixture of

l a r g e r and s m a l l e r p i e c e s , but Hershey (45) a p p l i e d a controlled

s h e a r i n g t o t h e T' DNA and i s o l a t e d w i t h an MAK column,

fragments w h i c h were a f r a c t i o n o f t h e s i z e of the original

molecules.

S u e o k a and Yamane (46) h a v e f r a c t i o n a t e d t R N A m o l e c u l e s

o n MAK columns, and demonstrated i t s heterogeneity. Sueoka

and C h e n g (50) found t h a t t h e r m a l l y denatured DNA-was e l u t e d

With s o l u t i o n s of a higher s a l t c o n c e n t r a t i o n t h a t those f o r

native D N A . They a l s o o b s e r v e d t h a t DNA molecules with a higher

G + C content tended t o be e l u t e d first. MAK columns a l s o a l l o w

t h e s e p a r a t i o n o f s o l u b l e and r i b o s o m a l RNA and e v e n 16S and

23S r i b o s o m a l RNA (47). From t h e s e e x p e r i m e n t s , Sueoka (47)

postulated that the o v e r a l l e l u t i o n pattern i s a r e s u l t of the

interplay of three e f f e c t s : the extent of hydrogen bonding,

b a s e c o m p o s i t i o n and m o l e c u l a r size.

The P h y s i c a l and C h e m i c a l Heterogeneity of DNA

These chromatographic t e c h n i q u e s have e n a b l e d the

i s o l a t i o n o f DNA relatively f r e e f r o m p r o t e i n and RNA contamina-

t i o n and f r a c t i o n a t i o n o f t h i s DNA into different components.

The m o l e c u l a r s i z e and b a s e c o m p o s i t i o n o f t h e s e f r a c t i o n s have

been determined, t o i n d i c a t e whether the f r a c t i o n s are fragments

o f homogenous DNA m o l e c u l e s , or whether they are i n f a c t

d i s t i n c t DNA molecules. In bacteriophage.:and some b a c t e r i a l DNA

 16

p r e p a r a t i o n s , t h e r e appears t o be one molecule o f DNA per c e l l

and the DNA can be e x t r a c t e d from p r e p a r a t i o n s as s m a l l e r

molecules o f uniform l e n g t h and composition (45). On the other

hand, p l a n t o r animal DNA c o n t a i n s molecules o f d i f f e r e n t s i z e s

and composition (48,49).

D e n s i t y g r a d i e n t e q u i l i b r a t i o n has been used t o separate

DNA molecules a c c o r d i n g t o s i z e and t o the percentage o f guanine

plus cytosine (G + C) . S c h i l d k r a u t e t al« (50) observed a d i r e c t

r e l a t i o n s h i p between the buoyant d e n s i t y (p) o f the DNA and i t s

G + C content as i n d i c a t e d i n the f o l l o w i n g equation:

p = 1.660 + 0.098 (G + C)

Takahash'i and Marmur (51) used t h i s e q u a t i o n to calculate

the percentage o f G + C o f DNA from a t r a n s d u c i n g phage o f B..

subtilis. These authors found t h a t t h i s DNA had a G + C content

o f 67%, whereas o t h e r methods had g i v e n a v a l u e o f 17.5%. This

anomoly was a t t r i b u t e d t o the f a c t t h a t the phage contained

u r a c i l i n s t e a d o f thymine.

T h i s procedure has proven t o be a v a l u a b l e method t o

study t h e p h y s i c a l o r chemical heterogeneity o f DNA. DNA from

most v e r t e b r a t e s p e c i e s e x i s t s as a s i n g l e band on c e n t r i f u g a t i o n .

With mouse and guinea p i g DNA, however, a s a t e l l i t e band

appears. T h i s minor band i n mouse DNA has a lower d e n s i t y than

the main band, w h i l e the r e v e r s e i s t r u e f o r t h e guinea p i g DNA

(52). In most h a l o b a c t e r i a , t h e r e i s o n l y a s i n g l e band o f DNA

w i t h a G + C content o f 55 - 64%. In one s t r a i n o f h a l o b a c t e r i a ,

 17

Joshi (53) observed t w o DNA species, one w h i c h comprises 20%

of the t o t a l DNA and has a G + C content of 58% a n d t h e other

which has a G + C content of 67%.

Intracellular differences i n DNA composition have been

indicated by t h i s method. In the plant Chlamydomonas, the

whole cell DNA contains 6% s a t e l l i t e DNA, whereas i n the

chloroplast, the s a t e l l i t e band is 39% o f the t o t a l chloroplast

DNA. As the c h l o r o p l a s t DNA is only 2% o f t h e t o t a l cellular

DNA, but contains 25% o f t h e t o t a l satellite DNA, Sager proposed

(54) that this satellite DNA m a y b e r e l a t e d to nonchromosomal

heredity factors which influence chloroplast division. This

theory is also supported by the o b s e r v a t i o n that i n aplastidic

algae/ there is o n l y o n e m a i n DNA b a n d w i t h a buoyant density

of 1.708, whereas i n corresponding green algae, there is a

m a i n DNA b a n d w i t h a density of 1.708 and a s a t e l l i t e band of

density 1.688. A lighter satellite band is also found i n a

mutant aplastidic strain (55) .

Denatured DNA of bacteriophage SPg h a s two components

of different buoyant densities; t h e H (heavy) strand has more

Pyrimidines than the L (light) strand and i s the only strand

which forms hybrids with t h e RNA synthesized by the host (56).

The thermal denaturation temperature, T ^ o f DNA also

reflects i t s G + C content. Early studies by Cosgrave and

Jordan (10) indicated that DNA h e a t e d i n a neutral salt

solution had p r o p e r t i e s differing from those of native DNA.

 18

Thomas (10) found t h a t the u l t r a v i o l e t a b s o r p t i o n i n c r e a s e s

above a c e r t a i n temperature. A t temperatures g r e a t e r than 80°C /

the v i s c o s i t y and a c t i v i t y o f t r a n s f o r m i n g DNA decreases as

well.

The temperature at which the s t r a n d s o f DNA s e p a r a t e

was found t o depend upon the i o n i c s t r e n g t h o f the s o l v e n t

and the r a t i o o f the amount o f adenine p l u s thymine t o quanine

p l u s c y t o s i n e (the base r a t i o ) o f the DNA (-51). Heavy m e t a l s ,

diamines and h i s t o n e s , which b i n d t o DNA, a l s o a f f e c t t h e T .

N a t u r a l l y the T^ a l s o depends upon the n a t u r e o f the DNA itself.

P o l y d i s p e r s i t y l e a d s t o a broadening o f the temperature

t r a n s i t i o n range (58) .

Marmur and Doty have expressed (59) t h e r e l a t i o n s h i p

between the T and the base c o m p o s i t i o n a s :

M w

T M = 69.3 + 0.4l(G + c)

where the T M i s the temperature o f the m i d p o i n t o f the t r a n s i t i o n

from double s t r a n d e d t o s i n g l e stranded DNA as measured by

hyperchromicity. T h i s e q u a t i o n a p p l i e s t o measurements taken

in standard s a l i n e c i t r a t e (0.15 M NaCl, 0.015 M c i t r a t e pH 7.0).

As hydrogen bonding between G and C i s much s t r o n g e r t h a n . t h a t

between A and T, the T^ i s thus h i g h e r i n DNA molecules w i t h

a h i g h e r p r o p o r t i o n o f G + C.

A t temperatures approximately 5°C above the T ,'the two

s t r a n d s o f the DNA h e l i x come a p a r t through Brownian movement.

I f the s o l u t i o n i s c o o l e d r a p i d l y , the two s t r a n d s remain

 19

separate, but i f the s o l u t i o n i s cooled slowly, specific

recombination of the two strands may o c c u r to restore the

double helix. This was d e m o n s t r a t e d by o b s e r v i n g changes i n

the buoyant density of pneumococcal DNA u p o n h e a t i n g a n d

cooling (60). Pneumococcal DNA i n i t s n a t i v e form has a density

of 1.700. When i t w a s d e n a t u r e d by heat and r a p i d l y cooled,

the single-strand material had a d e n s i t y of 1.716, b u t when i t

was slowly cooled, the strands recombined to give a helical

form with a density of 1.704. Recombination does not occur

readily with m a m m a l i a n - DNA a s i t tends t o be heterogenous i n

nature (60). — . .

Metabolic Heterogeneity

In a d d i t i o n t o p h y s i c a l and chemical differences, there

have been experiments t o show t h a t there may b e d i f f e r e n c e s in

metabolic activity and f u n c t i o n o f DNA. E a r l y experiments in

which the uptake of radioactive precursors into DNA w a s m e a s u r e d

indicated a preferential labelling of some o f t h e DNA f r a c t i o n s .

Bendich et a l . (28) fractionated DNA o f v a r i o u s rat tissues into

•

a soluble and i n s o l u b l e fraction i n 0.8% N a C l . The specific

activities were different i n the two f r a c t i o n s and these

differences varied with each tissue. The d i f f e r e n c e i n turnover

between the two f r a c t i o n s was a l s o indicative of metabolic

heterogeneity.

Frankel and Crampton (39) studied the metabolic activity

of DNA i n E h r l i c h ascites tumor c e l l s . T h e DNA w a s labelled

 20

14 * 3
with either C-formate or ' H-thymidine i n vivo and i n vitro.

The isolated DNA w a s then fractionated on an A m b e r l i t e 1RC-50

column. After short labelling periods, a DNA f r a c t i o n , which

was approximately 3% o f the total DNA, c o n t a i n e d up t o 11% of

the total radioactivity. The specific activity of the

remaining fractions varied greatly as well.

I n __ coli, the DNA o f untreated lysates is mainly found

in the particulate fraction which sediments at low centrifugal

forces. After a brief sonic o s c i l l a t i o n , , most of the DNA i s

recovered in the high speed supernatant, although a small

amount, which is rich i n newly synthesized DNA, i s not soluble

even after extensive sonic treatment. This treatment does not

'alter the d i s t r i b u t i o n of RNA ( 6 1 ) . DNA s y n t h e s i s , therefore

occurs predominantly i n a heavy particulate fraction and the

newly synthesized DNA i s especially resistant to sonic

degradation, possibly because it is bound to protein.

•Newly s y n t h e s i z e d DNA c a n be separated from the bulk of

the DNA b e c a u s e i t is bound to protein and t h e r e f o r e remains iri

the interphase after chloroform/isoamyl alcohol extraction of

nucleoprotein. The specific activity of the interphase DNA i s

13 times greater than the bulk DNA ( 6 2 ) . After addition of

unlabelled thymidine'to the incubation mixture in a chase •

experiment the specific activity of the interphase DNA d e c r e a s e s

while that of the remaining DNA i n c r e a s e s .

This "nascent" DNA a l s o differs in its physical state

from the "native" DNA. With cesium chloride centrifugation of

 21

a labelled E. coli DNA p r e p a r a t i o n , Rolfe (63) detected that the

DNA m o l e c u l e s i n the earliest stages of r e p l i c a t i o n were found

in two b a n d s , one heavier and one lighter than the principal

DNA b a n d . The DNA m o l e c u l e s of the heavier band had a collapsed

structure, i n which the subunits were dissociated, whereas the

light b a n d was v e r y unstable.

Frankel (64) was able to isolate replicative DNA f r o m T 2

phage infected bacteria by p r e v e n t i n g formation of phage

particles with an i n h i b i t o r of protein synthesis. T h i s DNA

had a h i g h e r molecular weight than normal phage DNA a n d had

properties of an i n c o m p l e t e double h e l i c a l structure.

From p l a n t tissues Sampson et a l . (65) obtained t w o DNA

fractions by chromatography o n a n MAK c o l u m n . One h a d a

5

molecular weight of 10 and a r a p i d rate of turnover. The

other, higher molecular weight fraction had a v e r y low rate of

turnover. The high molecular weight DNA h a d t h e same base

composition, irrespective of the tissue of origin, but the

composition of the low molecular weight DNA v a r i e d .

From c h l o r o p l a s t s of C h l o r e l l a , Iwamura and Kuwashima (66)

isolated t w o DNA s p e c i e s which differed in their buoyant

densities in CsCl. The lighter minor species was the metabolic

DNA a s i t was rapidly labelled with phosphate when t h e r e was

n o DNA s y n t h e s i s . The heavier major fraction is postulated to

be the genetic DNA. "The function of this metabolic species .

may be the synthesis of non-genetic DNA r e l a t e d to vigorous

energy-producing processes of the organelles" (66).

 Recent experiments indicate that n o t a l l DNA molecule-

are simultaneously replicating f r o m one end o f t h e m o l e c u l e L Q

the other. The p r o c e s s i s now thought t o be a semi-conservative

mechanism which i s s e q u e n t i a l a l o n g t h e chromosome. Initiation

and termination of replication occurs at well-defined sites o n

the chromosome ( 6 7 ) . This gives rise t o a number o f f o r k s ,

which c a n be d e t e c t e d by e l e c t r o n m i c r o s c o p y . T h e number o.f

f o r k s p e r chromosome d e p e n d s on t h e g r o w t h medium and c u l t u i o .

conditions. ( 6 8 ) , Mueller and K a j i w a r a ( 6 9 ) reported the

presence o f e a r l y and l a t e replicating DNA complexes in sync:( i r o n ou

cultures o f He L a c e l l s . They c o n c l u d e d that synthesis involves

discrete DNA u n i t s , which a r e made a t d i f f e r e n t times and a i , c

distributed non-randomly along t h e chromosomes. In e a r l y

labelling u n i t s , DNA replicates on a d d i t i o n o f t h y m i d i n e t o the

system which p r e v i o u s l y contained no t h y m i d i n e . The synthesis

o f b o t h new RNA and p r o t e i n a r e n e c e s s a r y for late replicating

DNA. This suggested to Mueller and K a j i w a r a ( 6 9 ) , that thei, G

are two d i f f e r e n t p h y s i c a l a s s o c i a t i o n s o f DNA w i t h i n the

c o m p l e x s t r u c t u r e o f mammalian chromosomes.

The new DNA has a h i g h e r affinity f o r MAK, a lower

sedimentation rate i n neutral or alkaline sucrose gradients

and a higher-sensitivity to alkaline degradation than nativ< }

DNA. ( 7 0 ) . The a b i l i t y o f CME-carbodiimide to bind to the ru ! W iy

synthesized DNA indicated to Salganik e t a l . , (71) t h a t ther.t.

molecules contain denatured regions. These small molecules

o f newly s y n t h e s i z e d DNA a c c u m u l a t e on n i a c i n starvation i n

bacteria (72). A f t e r a d d i t i o n o f n i a c i n , they"are integrated

 s t e p w i s e i n t o l a r g e r DNA m o l e c u l e s . I n summary, t h e new DNA,

s y n t h e s i z e d by p o l y m e r a s e s from p a r e n t a l DNA a n d n u c l e o t i d e

p r e c u r s o r s , appears i n t r a n s i e n t b u t m e t a b o l i c a l l y stable

r e p l i c a t i v e forms w h i c h a r e p r o g r e s s i v e l y j o i n e d t o t h e l o n g e r

chains (73) .

I t i s n o t known how t h e p r o c e s s o f DNA r e p l i c a t i o n i s

linked to c e l l division. I f i n i t i a t i o n and t e r m i n a t i o n o f c e l l

d i v i s i o n i s t r i g g e r e d by i n i t i a t i o n and t e r m i n a t i o n o f DNA

r e p l i c a t i o n , t h e h e t e r o g e n e i t y o f g e n e r a t i o n t i m e s may r e f l e c t

a heterogeneous r a t e o f DNA r e p l i c a t i o n ( 6 8 ) .

The P r e s e n t I n v e s t i g a t i o n

•Previous i n v e s t i g a t i o n s i n t h i s l a b o r a t o r y (14) have

i n d i c a t e d t h a t t h e DNA. w h i c h i s i s o l a t e d from r a t i n t e s t i n a l

mucosa y i e l d s f r a c t i o n s on chromatography on an MAK column

w h i c h d i f f e r i n base c o m p o s i t i o n and i n c o r p o r a t i o n o f a .

l a b e l l e d precusor. The m e t a b o l i c a c t i v i t y v/as d e t e r m i n e d by

m e a s u r i n g t h e u p t a k e o f r a d i o a c t i v e t h y m i d i n e i n vivo., because

t h y m i d i n e i s s p e c i f i c a l l y i n c o r p o r a t e d i n t o DNA w i t h o u t p r i o r

degradation. The a n i m a l s were i n j e c t e d w.ith H - t h y m i d i n e and

3

14 •

t w e n t y - f o u r h o u r s l a t e r , t h e y were i n j e c t e d w i t h C-thymidine.

Twenty o r f o r t y m i n u t e s a f t e r t h i s second i n j e c t i o n , t h e a n i m a l s

were s a c r i f i c e d and t h e DNA was i s o l a t e d from t h e i n t e s t i n a l

3 14
mucosa. The r a t i o o f H/ C was t a k e n as a measure' o f t h e
m e t a b o l i c a c t i v i t y o f t h e DNA f r a c t i o n s . A t 40 m i n u t e s , t h e r e
3 14 •
was no d i f f e r e n c e i n t h e H/ C among t h e DNA f r a c t i o n s from MAK
3 14 "
chromatography, whereas i n t h e 20 minute e x p e r i m e n t , t h e H/ C
increased i n the l a t e r DNA f r a c t i o n s from t h e chromatography

column.

I t was f e l t that t h e m e t a b o l i c d i f f e r e n c e s would be more

apparent i n experiments i n which injection o f t h e second

p r e c u r s o r was p e r f o r m e d shortly before s a c r i f i c e . Accordingly,

studies similar t o t h o s e above were done i n w h i c h the animals

14

were e x p o s e d t o the. C-thymidine f o r 5 o r 10 m i n u t e s . The base

c o m p o s i t i o n o f t h e DNA i n e a c h fraction o f t h e DNA peak was

determined f r o m ' i t s T value (59). Samples o f e a c h DNA fraction

3 14

were a l s o a s s a y e d f o r r a d i o a c t i v i t y and from t h i s , t h e H/ C

r a t i o s were calculated.
During the course of the i n v e s t i g a t i o n , i t became
3 14

apparent that t h e H/ C ratios calculated from f r a c t i o n s that

had been p r e v i o u s l y d e n a t u r e d by h e a t i n g d i f f e r e d from those

t h a t h a d riot b e e n exposed to this treatment. Studies carried

out i n d i c a t e d t h a t when t h e DNA s o l u t i o n was h e a t e d a n d c o o l e d ,

some n u c l e o t i d e m a t e r i a l p a s s e d t h r o u g h t h e d i a l y s i s b a g .
3 14

It had been assumed i n c o m p a r i n g t h e H/ C ratios of the

various fractions, that t h e DNA m o l e c u l e s a r e . u n i f o r m l y labelled

with, t r i t i u m . From t h i s , i t would be p r e d i c a t e d that the r a t i o

3

of t r i t i u m to o p t i c a l density ( H/O.D.) s h o u l d be c o n s t a n t f o r .

each f r a c t i o n . This expectation d i d not prove t o be t h e c a s e .

The ^H/O.D. r a t i o was c a l c u l a t e d f o r e a c h f r a c t i o n a n d f o u n d t o

vary i r r e g u l a r l y . I t i spossible, therefore, that during the
3

twenty-four hour exposure o f t h e t i s s u e to H-thymidine., there

is a t u r n o v e r i n t h e DNA, w h i c h w o u l d result i n an uneven

distribution of tritium i n t h e DNA m o l e c u l e s .

 25

G r o s s and Rabinowitz.(72) reported that i n r a t l i v e r there was

no further uptake of t r i t i a t e d thymidine into t h e DNA fraction

after three hours of exposure to the precursor. Therefore, a

set o f e x p e r i m e n t s was performed i n which the 3

H-thymidine was
14

injected into the rats three and a half hours before the C-

thymidine injection. In separate experiments, the animals were

sacrificed f i v e or 10 minutes a f t e r this second i n j e c t i o n . The

ratio of H/O.D. i n e a c h fraction f r o m MAK chromatography was

calculated. Under t h e s e c o n d i t i o n s , t h e r a t i o t e n d e d t o be
3 14
more, c o n s t a n t , b u t the p a t t e r n of H/ C . d i d not change.

One o f the disadvantages o f MAK chromatography i s t h a t

fractionation is a result o f the interplay of three factors:

d e g r e e .of h y d r o g e n b o n d i n g , b a s e c o m p o s i t i o n and molecular size

(47). Thus each f r a c t i o n could consist of widely different

DNA molecules. A more p r e c i s e way t o compare t h e DNA population,

is to fractionate the DNA preparation according to chain length

and then s u b f r a c t i o n a t e each f r a c t i o n according to base

composition, as d e s c r i b e d by Cerny e t a l , ( 4 0 ) . The DNA

p r e p a r a t i o n was hydrolyzed with diphenylamine-formic acid and

t h e h y d r o l y s a t e was chromatographed according to chain length

on a DEAE-cellulose column. Six to eight pyrimidine isostichs

were o b t a i n e d i n e a c h e x p e r i m e n t upon e l u t i o n of the hydrolysate

3 14
from the column. The radioactivity due to H and C was
determined f o r each i s o s t i c h . In the 5 minute experiment the
3 -•
radioactivity due to H- was l o c a t e d i n the p y r i m i d i n e isostichs
14

V I and V I I I ; than due to C was present mainly i n the isostichs

I I and V I I I . . In the 10 m i n u t e e x p e r i m e n t on the other hand,

 26

3 14

most o f t h e r a d i o a c t i v i t y due to both H and C was recovered

in the f i f t h isostich.

The e x p e r i m e n t a l technique o f Cerny e t a_l. ( 4 0 ) has great

possibilities i n d e t e r m i n i n g t o which base the radioactive

percursor is preferentially attached as w e l l as i n d e t e r m i n i n g

the base sequence of nucleic acids.

METHODS AND MATERIALS

1. Administration of Labelled Material

. The labelled precursor materials used i n these experiments

were m e t h y l - ^ H - t h y m i d i n e , p u r c h a s e d f r o m New England Nuclear

1 4

Corporation and 2- C-thymidine, obtained from the Radiochemical

C e n t r e , Amersham, E n g l a n d . I n e a c h e x p e r i m e n t , two male W i s t a r

r a t s w e i g h i n g a p p r o x i m a t e l y 200 mg. e a c h were u s e d . Each animal

was a n a e s t h e t i z e d w i t h e t h e r and injected through the t a i l vein

with 0.5 ml o f a p h y s i o l o g i c a l saline solution containing 0.1

m C i o f ^H-thymidine and 8.2 jjaaoles of unlabelled precursor.

Twenty-four hours after this injection, each animal r e c e i v e d i n

t h e same manner 0 . 5 ml o f s a l i n e solution containing 0.03 mCi
1 4

of C-thymidine and 8.2 jumoles o f t h e u n l a b e l l e d nucleoside.

Five or t e n minutes after the second i n j e c t i o n , the animal was

s t u n n e d by a b l o w on t h e head and d e c a p i t a t e d . The intestine

was removed f o r i s o l a t i o n o f DNA as d e s c r i b e d below.

2. Isolation o f DNA f r o m Rat I n t e s t i n a l Mucosa

T h i s p r o c e d u r e was b a s e d on t h a t o r i g i n a l l y described by

Colter et a l (27). % After removal o f the s m a l l intestine, i t was

 27

cut into small segments. Each segment was f l u s h e d free of its

contents with chilled saline-Versene solution (l.OMNaCl, O.02M

KH P0 2 4 and 0.01 M.EDTA), which i n h i b i t s DNAse. I . The segments

were c u t l o n g i t u d i n a l l y and placed mucosal side upwards on a

glass plate. The mucosa was removed by s c r a p i n g t h e edge o f a

microscope slide and placed i n a beaker filled with liquid

nitrogen. To 5 g o f the frozen tissue were added 45 m l . o f the

saline-Versene solution, 2.5 m l . of 10% s o d i u m deoxycholate

solution a n d 50 m l . o f w a t e r - s a t u r a t e d phenol. The l a t t e r was

prepared b y a d d i n g 25 m l . o f glass d i s t i l l e d water to 75 g o f

redistilled phenol. The m i x t u r e was h o m o g e n i z e d i n a Servall

Omni-mixer f o r 20 m i n u t e s at the 20 v o l t setting of. t h e V a r i a c

transformer. The e m u l s i o n w h i c h f o r m e d was c e n t r i f u g e d for 10

minutes at 27,000 x g i n a Servall refrigerated centrifuge. The

upper aqueous layer containing t h e DNA w a s c a r e f u l l y removed.

An equal volume of water-saturated p h e n o l was added t o t h e DNA

solution and the m i x t u r e was s h a k e n f o r 5 minutes. The p h e n o l

and aqueous phases were separated by c e n t r i f u g i n g at 6000 r p m

(4340 x g ^ ) f o r 10 m i n u t e s i n the S e r v a l l refrigerated centrifuge.

The upper layer was removed and e x t r a c t e d a second time with

phenol as d e s c r i b e d above.

Traces of phenol were removed by repeated extractions with

ether, w h i c h was i n t u r n removed by b u b b l i n g n i t r o g e n through

the solution. T h e DNA s o l u t i o n w a s d i a l y z e d e x t e n s i v e l y against

a saline-citrate solution (O.Ol'.M s o d i u m c i t r a t e , 0.05 M sodium

chloride and 0.05M phosphate.) pH 6.3 and then stored at -15°C.

Prior to i t s use, the d i a l y s i s t u b i n g was washed with successive

 28

c y c l e s o f d i s t i l l e d w a t e r and 0 . 5 N NaOH. The t u b i n g was t h e n -~

washed f r e e o f a l k a l i and s t o r e d i n d i s t i l l e d water a t - 4 0 ° C .

T h i s p r o c e d u r e removes u l t r a v i o l e t a b s o r b i n g m a t e r i a l from t h e

tubing.

3. Chromatography o f t h e DNA S o l u t i o n on M e t h y l a t e d - A l b u m i n
Kieselguhr ;

T h i s t y p e o f chromatography was f i r s t d e s c r i b e d by Lerman

(41) and i t s use "was d e v e l o p e d by M a n d e j l and Hershey (42). In

t h i s method, a 'column c o n s i s t i n g o f l a y e r s o f m e t h y l a t e d - a l b u m i n

a r e used. The k i e s e l g u h r used was t h e m a t e r i a l s o l d as C e l i t e

by J o h n s - M a n s v i l l e Company. The m e t h y l a t e d - a l b u m i n had been

p r e v i o u s l y p r e p a r e d i n t h i s l a b o r a t o r y by C. M e z e i by t h e method

o f M a n d e l l and Hershey (42) .

The b u f f e r s used i n p r e p a r i n g t h e columns a r e l i s t e d as

follows:

B u f f e r No.

1. 0.05.M sodium c h l o r i d e i n 0.051M phosphate b u f f e r pH 6 .

2. 0.1IW sodiumc h l o r i d e i n 0 . 0 5 7 4 phosphate b u f f e r pH 6 . 7
3. 0.4.M sodiumc h l o r i d e i n 0 . 0 5 M phosphate b u f f e r pH 6 . 7
4. 1.5.Msodium c h l o r i d e i n 0 : 0 5 J H phosphate b u f f e r pH 6 . 7

a) Preparation o f t h e P r o t e i n .Coated Celite

A s u s p e n s i o n o f 20 g o f C e l i t e i n 1 0 0 m l . o f b u f f e r

2 was b o i l e d t o e x p e l a i r and t h e n c o o l e d t o room temp-

erature. F i v e m l . o f a 1% s o l u t i o n o f m e t h y l a t e d - a l b u m i n

i n waterwere- added, w i t h s t i r r i n g , f o l l o w e d by 20 m l . o f

b u f f e r 2. The c h r o m a t o g r a p h i c column was p r e p a r e d i n a

2 x 2 5 cm. column and was s u p p o r t e d by a s i n t e r e d g l a s s

 29

disk a t the d e l i v e r y end. Before adding the s l u r r y , a

small layer o f W h a t m a n . c e l l u l o s e powder was layered over the

sintered disk. The column was packed a f t e r addition of

each p o r t i o n under a pressure of 3 p . s . i . The column

was washed w i t h 300 ml. of b u f f e r 4, a f t e r w h i c h the

c o n t e n t s o f t h e . c o l u m n was" removed, suspended i n 125 ml.

of buffer 3 and stored at -40°C.

b) P a c k i n g o f t h e MAK Column

For the f i r s t layer, 8 g . o f C e l i t e were suspended

in 40 m l . o f b u f f e r 2 and the suspension-was b o i l e d and

cooled. Two m l . o f 1% m e t h y l a t e d a l b u m i n s o l u t i o n were

added w i t h stirring and t h e n 15 m l . o f b u f f e r 2. The

final s u s p e n s i o n was added i n 10 m l . p o r t i o n s in a 2 x 25

cm. c h r o m a t o g r a p h i c column. The c o l u m n was packed under

a pressure of 3 p.s.i. after each a d d i t i o n of s l u r r y .

Any s u s p e n s i o n r e m a i n i n g on t h e s i d e s o f t h e column was

washed down w i t h b u f f e r 1 to avoid contaminating the

second layer.

Twenty ml. o f the p r o t e i n - c o a t e d C e l i t e were added

to a s u s p e n s i o n o f 12 g C e l i t e i n 80 m l . of buffer 3,

w h i c h had been b o i l e d and c o o l e d . The second l a y e r of

the c o l u m n was formed from t h i s slurry i n a similar .

manner t o t h a t f o r the f i r s t layer. The final layer was

formed from a b o i l e d and c o o l e d s l u r r y o f 1 g. C e l i t e i n

" ; .10 ml. b u f f e r 3. The c o l u m n was washed'with'buffer 1 *

until t h e a b s o r b a n c e a t 260 nm o f t h e e f f l u e n t was -zero.•

 30

and the c o n d u c t i v i t y o f the e f f l u e n t was the same as

t h a t o f the eluent.

A l l s p e c t r o p h o t o m e t r i c measurements were done on a

G i l f o r d model 2000. Recording Spectrophotometer

equipped w i t h a t h e r m o s t a t a b l e c e l l compartment. The

c o n d u c t i v i t i e s were measured w i t h a Radiometer conductivity

meter. A s t a n d a r d c u r v e o f sodium c h l o r i d e c o n c e n t r a t i o n .

i n the phosphate b u f f e r v e r s u s c o n d u c t i v i t y was drawn and

from t h i s c u r v e , the c o n c e n t r a t i o n o f N a C l i n a-n unknown

s o l u t i o n c o u l d be determined.
l
c) F r a c t i o n a t i o n o f the DNA S o l u t i o n by MAK Chromatography

- A p o r t i o n o f the DNA solution containing approximately

40 absorbance u n i t s * was a p p l i e d t o the, column under a

p r e s s u r e o f 3 p . s . i . A f t e r the sample, had been absorbed

on the column, e l u t i o n was c a r r i e d out w i t h a l i n e a r

gradient s o l u t i o n o f sodium c h l o r i d e e s t a b l i s h e d by u s i n g .

400 ml. o f b u f f e r 1 i n the m i x i n g chamber and 400 ml. of

b u f f e r 4 i n the r e s e r v o i r . A B u c h l e r micro-pump was used

t o o b t a i n a f l o w r a t e o f . a b o u t 25-30 ml. per hour. Five

ml. f r a c t i o n s o f e f f l u e n t were c o l l e c t e d and the

absorbance o f each a t 260 nm was measured. The sodium

chloride concentration o f each f r a c t i o n was also

determined.

4. Thermal D e n a t u r a t i o n Curves

An absorbance u n i t i s the. amount o f n u c l e i c a c i d per m i l l i l i t e r

of s o l u t i o n w h i c h has an o p t i c a l d e n s i t y o f 1 f o r a l i g h t p a t h
of 1 cm (14). ;
 31

The base c o n t e n t o f each f r a c t i o n was d e t e r m i n e d by t h e

method o f Marmur and Doty (59) . T h i s method i s based on t h e i r

f i n d i n g t h a t t h e temperature a t which'the two s t r a n d s o f a DNA

m o l e c u l e s e p a r a t e depends on t h e amount o f guanine p l u s c y t o s i n e

i n the molecule. The t h e r m a l d e n a t u r a t i o n c u r v e s were determined

as d e s c r i b e d by Marmur and Doty ( 5 9 ) .

In order t o study the thermal d e n a t u r a t i o n curves o f the

DNA f r a c t i o n s e l u t e d from t h e MAK column, t h e s e were f i r s t

d i a l y z e d against standard s a l i n e - c i t r a t e s o l u t i o n (0.15 >M N a C l -

0.0I'5.:M Na c i t r a t e pH 7.0). The i n c r e a s e i n absorbance with

temperature o f t h e d i a l y z e d DNA f r a c t i o n s was r e c o r d e d a u t o -

m a t i c a l l y u s i n g a G i l f o r d Model 2000 r e c o r d i n g . s p e c t r o p h o t o m e t e r ,

which.was equipped t o r e c o r d changes i n o p t i c a l d e n s i t y w i t h

temperature. The t e m p e r a t u r e i n t h e c u v e t t e chamber c o u l d be

r a i s e d a t a u n i f o r m r a t e w h i l e b e i n g r e c o r d e d and t h e o p t i c a l

d e n s i t y c o u l d be r e c o r d e d a u t o m a t i c a l l y . The t e m p e r a t u r e

corresponding t o t h e mid-point o f t h e hyperchromic s h i f t was

t a k e n t o be t h e T„ v a l u e .
M

5. R a d i o a c t i v e Counting Procedures •

The method was counted i n a P a c k a r d model 314A T r i - C a r b

li-quid-scintillator spectrophotometer. The a n a l y s i s mode s w i t c h

was i n " s p l i t " and number "2" p o s i t i o n (75). Counting of the

d o u b l y - l a b e l l e d sample was c a r r i e d o u t i n a system used by C. M e z e i

(14.) made up as f o l l o w s . O n e - h a l f m l . o f t h e DNA s o l u t i o n was

p l a c e d i n a c o u n t i n g v i a l and t o t h i s was added 0.5 m l . o f hyamine

h y d r o x i d e p r e p a r e d by t h e method o f ..Eisenberg •."( 76 )•,: . '• _ 4-. 0 m l .

 32

of absolute ethanol'and 5.0 ml. o f a s c i n t i l l a t o r solution. The

latter c o n s i s t e d o f 0 . 6 3 % P P O and 0 . 0 2 6 2 % POPOP in redistilled

toluene.

It i s p o s s i b l e t o assay f o r both t r i t i u m and c a r b o n - 14

in a d o u b l e - l a b e l l e d compound due t o t h e e f f e c t i v e n e s s o f liquid

scintillators f o r counting weak b e t a - e m i t t i n g isotopes and t h e

difference i n the energies of the beta particles o f t h e two

3

isotopes (77). The maximum b e t a energy f o r H i s 0.018 Mev and

for 0.155 Mev a n d t h e two i s o t o p e s may be c o u n t e d i n one

operation by p r o p e r s e l e c t i o n o f h i g h voltage and d i s c r i m i n a t o r

settings (76) . T h e two c h a n n e l s f o r pulse-height discrimination

are obtained b y means o f d i s c r i m i n a t o r c o n t r o l s A A ; B and C . 1

All pulses o f e n e r g y between A A 1

t o B are recorded on t h e r e d

s c a l e r and .pulses from B t o C a r e recorded on t h e g r e e n s c a l e r .

The counting rate of a given standard was d e t e r m i n e d a t t h e

d i f f e r e n t high voltage settings. The s t a n d a r d solutions consisted

of t r i t i a t e d water c o n t a i n i n g 98,000 cpm and a s t a n d a r d solution

o f Na^CO^ c o n t a i n i n g 44,000 cpm (78). To o b t a i n t h e o p t i m a l

setting, a s e r i e s o f g r a p h s was c o n s t r u c t e d showing t h e c o u n t i n g

rate as a f u n c t i o n o f t h e h i g h voltage setting at different

discriminator settings. T h e s e g r a p h s a r e shown i n F i g u r e s 3

and. 4 .
14
A standard s o l u t i o n containing both Na 2 CO^ (44,000 dpm)
3
and E^O (98,000 dpm) was p r e p a r e d and c o u n t e d at the various
3 14

settings. The r a d i o a c t i v i t y due t o H or C was c a l c u l a t e d

according t o t h e d i s c r i m i n a t o r - r a t i o method. T h i s method i s

described by O k i t a e t a l , ( 7 7 ) , who d e r i v e d the following

3* CaJ
 3 3

F i g . 3. I n t e g r a l d i s c r i m i n a t o r b i a s c u r v e s f o r standard H ( H,jO)

and ( N^^COj ) o b t a i n e d from the red s c a l e r o f a

Packard 314- AX L i q u i d S c i n t i l l a t o r Spectrophotometer.

33 (a)
 3
!4

F i g . 4. I n t e g r a l d i s c r i m i n a t o r bias curves f o r standard H and C

determined on the green s c a l e r . The curves represent the

average of the determination of s i x d i f f e r e n t window s e t t i n g s .

 3U<

1 2 3 4
High- Voltage Sett
 35

3 14
equations' f o r obtaining H and C cpm from doubly-labelled
compounds:

b N
l -
2 N

3„ J b - a
H dpm = j
red scaler H efficiency factor = 0.034

b(N 2 - a^)

14^, , b - a
C dpm = ^4
green s c a l e r C efficiency factor i s 0.236

where = n e t cpm on r e d s c a l e r

N 2 = n e t cpm o n g r e e n scaler

3
-_ n e t cpm o f _ H on
. green s c a l e _r
a

:
n e t cpm o f H on r e d s c a l e r

14
, _ n e t cpm o f C on g r e e n scaler
14
net cpm o f C on r e d s c a l e r

From F i g u r e s 3 and 4 , the following constants were

calculated:

a = .04, b = 1.1/ the red scaler 3

H efficiency
14

factor = 0.34 and t h e g r e e n s c a l e r C efficiency factor

is .236.
3 14

The percent recoveries of H and C i n the standard

solution were c a l c u l a t e d to find which s e t t i n g gave t h e h i g h e s t

recovery f o r both isotopes. These c a l c u l a t i o n s indicated

that a high voltage setting of 6, corresponding to 1025 volts,

and discriminator settings of 10:50:90 were t h e b e s t f o r

simultaneous counting of both isotopes.

 36

EXPERIMENTAL

1. ' A d m i n i s t r a t i o n o f L a b e l l e d T h y m i d i n e Precursors

Earlier investigations b y C. Mezei (14) h a d suggested

the p o s s i b i l i t y t h a t r a t i n t e s t i n a l m u c o s a may contain d i s t i n c t

DNA fractions which d i f f e r i n t h e i r metabolic a c t i v i t y . The

incorporation of ^H-thymidine, which i s a s p e c i f i c precursor

f o r DNA, was u s e d a s an i n d i c a t i o n of metabolic a c t i v i t y .

3 . >
The H - l a b e l l e d DNA was fractionated on an MAK c o l u m n and the

s p e c i f i c a c t i v i t i e s o f .each f r a c t i o n w e r e d e t e r m i n e d . One of

the difficulties of s i n g l e - l a b e l l i n g experiments i s the error

due t o i s o l a t i o n b e c a u s e i d e n t i c a l - f r a c t i o n s - can-not'-'be. i s o i - a t e d i n

every experiment. The difficulty i n interpretation as a result

of v a r i a t i o n i n DNA isolated from experiment to experiment was

overcome by t h e s e n s i t i v e method o f d o u b l e l a b e l l i n g . To use

t h i s t e c h n i q u e , r a t s were i n j e c t e d f i r s t with tritiated

• 14
t h y m i d i n e and t h e n i n j e c t e d with the C-labelled precursor.
I t was assumed (14) t h a t d u r i n g t h e 24 h o u r s t h e DNA would be
14
u n i f o r m l y l a b e l l e d w i t h t r i t i u m and t h e u s e o f C-thymidine
would l a b e l newly f o r m e d DNA. The DNA.was f r a c t i o n a t e d and
3 14
the r a d i o a c t i v i t y o f e a c h f r a c t i o n was determined. The H/ C
r a t i o was constant i n the experiment i n which the r a t s were
14
s t u n n e d and k i l l e d f o r t y minutes a f t e r the i n j e c t i o n o f C-
thymidine. This finding s u g g e s t e d t h a t t h e o l d and new
14
molecules are u n i f o r m l y l a b e l l e d w i t h C. A t a s h o r t e r time
3 14

i n t e r v a l of twenty minutes, though, d i f f e r e n c e s i n the H/ "C

r a t i o i n the f r a c t i o n s f r o m t h e MAK column d i d . appear. The

fractions eluted w i t h an e l u e n t a t a h i g h c o n c e n t r a t i o n of
 37

sodium c h l o r i d e had h i g h e r 3
H/ 1 4
C ratios. These l a t e r fractions

were shown by T ^ m e a s u r e m e n t s and C s C l b u o y a n t d e n s i t y centrif-

ugation t o c o n t a i n a lower G + C content.

In t h e p r e s e n t investigation, a series of similar

experiments was carried out except that the animals were

14

sacrificed 5 minutes- a f t e r injection of C-thymidme. A 20

m i n u t e e x p e r i m e n t was also carried o u t t o check t h e r e s u l t s

obtained p r e v i o u s l y (14).

2. C h a r a c t e r i z a t i o n of the I s o l a t e d DNA

In mammalian cells, deoxyribonucleic acids are i n the

form o f a DNA-histone complex located within the n u c l e i , DNA

can be " i s o l a t e d b y m e c h a n i c a l l y d i s r u p t i n g the c e l l u l a r and

n u c l e a r membranes a n d r e m o v i n g t h e b o u n d p r o t e i n w i t h phenol

or isoamyl a l c o h o l / c h l o r o f o r m (79) . High molecular weight DNA

in i t s native state i s very susceptible to denaturation or

degradation to smaller molecules by c h e m i c a l or shearing

processes. For this reason,.it i s d i f f i c u l t to obtain a

quantitative yield o f p u r e , u n d e r g r a d e d DNA molecules.

Because o f t h e success i n this l a b o r a t o r y b y C. M e z e i (7 8)

with t h e i s o l a t i o n m e t h o d o f C o l t e r e t al_. (27) f t h i s method was

selected f o r the following investigations.

,A sample o f t h e i s o l a t e d DNA contained 14.4% n i t r o g e n ,

and 9.6% p h o s p h o r u s , g i v i n g an a t o m i c N/P ratio o f 3.32 which

agreed with values r e p o r t e d by C h a r g a f f (80) f o r a s e r i e s o f

high molecular weight DNA s.1

 38

3. Chromatography of the Isolated DNA o n M e t h y l a t e d - A l b u m i n

K i e s e l g u h r Columns •

A prerequisite for the study of metabolic differences in

a DNA p o p u l a t i o n i s a suitable technique which permits the

fractionation of these molecules. As s t a t e d earlier,

Sueoka a n f j Cheng (47) found that MAK c h r o m a t o g r a p h y w i l l enable

the fractionation of DNA m o l e c u l e s according to base

composition, molecular size, and degree of hydrogen bonding.

Unlike ECTEOLA-cellulose chromatography (78) , the chromatography

of DNA o n MAK y i e l d s a' s h a r p peak of deoxyribose - containing

material which is eluted with a sodium c h l o r i d e concentration

of 0.5 . ;
to 0.6M. Rechromatography of this material again

gives a single peak at the same c o n c e n t r a t i o n of eluting buffer.

Chromatography of other samples of the s a m e DNA s o l u t i o n give

similar fractionation profiles. Because of this reproducibility

and h i g h e r p o s s i b l i t y of dealing with the s a m e DNA f r o m

experiment to experiment, MAK c h r o m a t o g r a p h y has been a widely

used tool i n i n v e s t i g a t i n g DNA.

• Within the main peak eluted from the MAK c o l u m n , is

approximately 95% of the total deoxyribose material detected

in the eluate as measured by t h e Dische test (14). Other

smaller peaks of ultraviolet-absorbing material are eluted at

lower s a l t concentrations. These peaks, however, do not

contain deoxyribose or radioactive material. The peak which is

eluted just before the DNA p e a k , i s thought to be RNA because:

it is not present if the sample is treated with RNAsebefore it

is applied to the column (81). The other peaks contain some

 39

unidentified u l t r a v i o l e t - a b s o r b i n g m a t e r i a l , which remains i n

t h e DNA solution even after extensive dialysis.

Measurements o f b u o y a n t d e n s i t i e s by o t h e r w o r k e r s (14)

and the< m e a s u r e m e n t s i n t h e p r e s e n t work h a v e shown t h a t t h e

f r a c t i o n s making Up the s i n g l e peak o f DNA differ i n base

composition i n a graded manner. I n F i g u r e 5 c a n be seen the

single l a r g e peak o f DNA m a t e r i a l obtained.by chromatography

of the i s o l a t e d rat intestinal m u c o s a l DNA on an MAK column.

The fractionation p a t t e r n was not s i g n i f i c a n t l y altered when

t h e DNA had been s t o r e d a t -15°C f o r s e v e r a l months.

4. Thermal D e n a t u r a t i o n Curves

'Chemical behaviour among t h e DNA fractions obtained by

MAK chromatography w i l l vary with base c o m p o s i t i o n . A direct

technique f o r determining the base r a t i o i s to hydrolyze the

DNA sample liberating t h e f r e e b a s e s w h i c h c a n be separated by

paper, c h r o m a t o g r a p h y . Spectrophotometric analysis of the

ultraviolet-absorbing s p o t s on t h e c h r o m a t o g r a m will give the

quantitative amounts o f e a c h b a s e p r e s e n t . T h i s method r e q u i r e s

l a r g e r amounts o f DNA than those, i n e a c h f r a c t i o n obtained by

MAK chromatography. For this reason the s i m p l e r indirect

a p p r o a c h o f S . c h i l d k r a u t , Marmur and D o t y (50) was used. They

observed t h a t t h e b u o y a n t d e n s i t y o f a DNA molecule depends on

its G + C content and t h e r e f o r e , t h e y formulated the f o l l o w i n g

equation to express this relationship.

p = 1.660 + 0.098 ( G -F C) where p i s

t h e b u o y a n t d e n s i t y o f t h e DNA sample and i s d e t e r m i n e d by

• v • • .. • • "
 to. 8

40.7
/

DNApeak

[DM

L0.3 co

A -0.1
Si

ru
•0.1

10 20 30 HO . . so +
• 60 70 ,
Tube No.
F i g . 5. Chromatography of DNA from r a t i n t e s t i n a l mucosa on an MAK column. E l u t i o n was c a r r i e d out with a

eradient of NaCl i n 0 . 0 5 M. DhosDhate - DH 6.7

 4J,.

reference t o a DNA m o l e c u l e o f known d e n s i t y . The b u o y a n t

density a l s o d e p e n d s upon t h e m o l e c u l a r size o f t h e DNA and

will vary i f unnatural bases are present (50).

The G+C content c a n a l s o be c a l c u l a t e d f r o m t h e t h e r m a l

denaturation temperature ( T M ) a c c o r d i n g t o Marmur and D o t y ( 5 9 ) .

--The-behaviour .of....a-DNA ..molecule o n d e n a t u r a t i o n by h e a t i n g i n

a dilute a q u e o u s s o l u t i o n when i t u n d e r g o e s s t r a n d separation

is related t o i t s base composition. Since the hydrogen

b o n d i n g ' b e t w e e n G.and.C i a : s t r o n g e r t h a n t h a t between A

and T, t h e T M i s higher i n DNA's w i t h a higher proportion of

G a n d C.

At temperatures approaching the melting temperature,

the two s t r a n d s o f t h e DNA h e l i x come a p a r t t h r o u g h Brownian

-movement and c h a r g e r e p u l s i o n . I f the s o l u t i o n i s cooled

rapidly, t h e two s t r a n d s remain separate, but i f they are cooled

slowly, s p e c i f i c recombination o f t h e 2 s t r a n d s "may o c c u r t o

restore the double h e l i x . Evidence f o r s t r a n d s e p a r a t i o n on

d e n a t u r a t i o n has been p r o v i d e d by p h y s i c a l , chemical and

biological experiments (82).

A combination of light scattering, v i s c o s i t y , and

sedimentation c o e f f i c i e n t measurements i n d i c a t e d t h a t t h e

molecular w e i g h t o f t h e DNA p a r t i c l e s decreased by h a l f

following denaturation and r a p i d c o o l i n g . I f t h e DNA molecules

labelled with i 4
N were m i x e d w i t h ^ 5
N - l a b e l l e d DNA molecules
 42

of the same o r i g i n , h e a t e d and s l o w l y c o o l e d , hybrid DNA

14
molecules, i n w h i c h one strand is labelled with N and the
15
other with N, c o u l d be detected by C s C l density centrifugation.

Biological activity, w h i c h depends on the presence of

double stranded DNA, such as the transforming property of

certain bacterial DNA's, is lost on h e a t i n g (60). On the

other hand, the immunological reaction of DNA increases on

denaturation as this is a property of single stranded DNA (10).

Strand separation can be detected by the increase in

absorption at 260 nm o r decrease in viscosity of the DNA

solution. The hyperchromic shift observed during this

process results from the d i s r u p t i o n of the characteristic

stacking of the bases maintained in native DNA as a result of

hydrogen bonding. If it is assumed that the increase in

ultraviolet absorption is proportional to the helix content,

the hyperchromicity would seem t o be a valid measure of the

extent of d i s r u p t i o n of helical regions which contain at least

7 or.8 residues (10).

A typical thermal denaturation curve -

of the DNA isolated

in the present work i s shown i n F i g u r e 6 . In Table I are

shown, t h e T^ v a l u e s for the fractions comprising the DNA peak

obtained b y MAK chromatography of DNA isolated in these

experiments. While the T s M varied slightly from experiment

to experiment, they a l l f o l l o w e d the same p a t t e r n . From the

table i t can be seen that the fractions with a higher G + C

content were eluted first from the MAK c o l u m n " a n d t h e r e was a

 4^.

lr s s I
1
tuu 0 9 2 IB '0*0
F i g . 6. E f f e c t of increasing temperature on the o p t i c a l density of

doubly-labelled r ? t . i n t e s t i n a l mucosal DNA.

 44

gradual decrease i n the G + C content with increasing fraction

number These findings are s i m i l a r to those reported by

Sueoka and Cheng (47) and M e z e i and Zbarsky (14) . 'I h e s e

findings suggest that there a r e s e v e r a l DNA s p e c i e s which

differ in t h e i r base composition. <_:heng a n d Sueoka (83)

confirmed these findings by density centrifugation.

Fraction Initial Final Change T

No. O.D. O.D. in O.D. _M % (G + C )

1 0.312 0.365 0.053 91.4 53.9

2 0.432 0.577 0.14.5 91.0 52.9
3 0.641 0 . 853 0.212 89.6 49.5
4 1.160 1.414 0.254 8 8.6 47.0
5 0.667 0.927 0.260 88.0 45.6
6 0.791 1.056 0.265 87.4 44.1
7 0.661 0.780 0.119 86.8 42.6
8 0.352 0.428 0.076 86.7 42.4
9 0.243 0.261 0.022 86.7 42.4

Table I - T., v a l u e s and G - C c o n t e n t of separate

M
fractions o f main peak after MAK c h r o m a t o g r a p h y o f DNA f r o m

intestinal mucosa.

5. Radioactive C o u n t i n g o f D o u b l y - L a b e l l e d DNA

As p o i n t e d o u t e a r l i e r , the difference i n uptake of a

radioactive precursor between t w o DNA f r a c t i o n s might

indicate a difference i n metabolic activity. The technique

bf l a b e l l i n g the molecules with two i s o t o p e s provides a very

sensitive method for detecting these differences. The r a d i o -

3 14

activity due t o H and C was c a l c u l a t e d f o r each DNA f r a c t i o n

f r o m t h e MAK c o l u m n b y t h e d i s c r i m a t o r r a t i o . m e t h o d of Okita-.

et at (77) .
44 [a)
Fig. 7. R a t i o o f ^ H/ i n f r a c t i o n s comprising the DNA pe^k eluted -by

.chromatography of d o u b l y - l a b e l l e d mucosal PNA, which h?d been

3
pllowed t o incorporate H f o r 24- hours, on MAK columns.
Each fraction is,one tube . The first tube i n the DNA

peak i s c a l l e d fraction no. 1.

 46 . • .

14
Figure 7 indicates how t h e r a t i o s i n which C-
3

thymidine was g i v e n 24 h o u r s after H-thymidine vary. In

experiments w h e r e DNA was i s o l a t e d 20 m i n u t e s a f t e r administra-

tion o f ^ C-thymidine
4
to rats, (A) t h e H / ^ C r a t i o
3 4
increased

in the l a t e r fractions i n agreement w i t h results obtained by

Mezei and Zbarsky ( 1 4 ) .

In experiment (B) where DNA was i s o l a t e d 5 minutes

after a d m i n i s t r a t i o n o f ^"^C-thymidine, curve o f the H/^ C 3 4

r a t i o versus fraction number consistently showed a maximum

of fraction 4 o r 5. This finding might suggest that there

a r e two m e t a b o l i c a l l y a c t i v e fractions, one e l u t e d a t t h e

beginning o f t h e DNA p e a k a n d t h e o t h e r a t t h e end. However,

as shown i n T a b l e I I i n the l a t e r fractions, the radioactivity

due t o H d e c r e a s e d
3
considerably, while the counts due t o
14
C and t h e o p t i c a l d e n s i t y o f t h e DNA m o l e c u l e s decreased
only slightly. T h i s o b s e r v a t i o n suggested t h a t perhaps the
3

DNA m o l e c u l e s are not uniformly labelled with H. In order

to test this possibility, the r a t i o of the H 3

radioactivity/

optical d e n s i t y was c a l c u l a t e d f o r each f r a c t i o n . The

results are l i s t e d i n Table I I .

3

Table I I - The r a t i o o f r a d i o a c t i v i t y due t o H/optical

density o f t h e DNA f r a c t i o n s obtained from MAK chromatography

in experiments i n w h i c h H - t h y m i d i n e was a d m i n i s t e r e d
3
to rats
14
24 h o u r s b e f o r e the injection of C-thymidine.
 47

DNA Fraction 3 3
Number H c/m P.P./ml. H/O.D.
1 76.8 1.910 40.2
2 9.41 2.960 3.1
3 2396.5 2.711 884
4 4359 1.845 2362
5 ' 4638 2.274 2040
6 4274 1.917 2660
7 4185 1.594 2546
8 4144 1.678 2347
9 4074 1.080 3772
10 4062 1.222 3324
11 4035 1.158 3847
12 4038 .660 6118

I t i s obvious from these c a l c u l a t i o n s , that H i s not

uniformly d i s t r i b u t e d throughout the DNA molecules. The

v a r i a t i o n was not regular. A possible explanation f o r t h i s

f i n d i n g i s that during the 24 hour incubation period, some of

the DNA may be degraded as the rate of turnover of c e l l s i n

..the , i n t e s t i n a l mucosa i s very high (84). Furthermore, the

experiments of Gross and Rabinowitz (74) indicated t h a t no

further uptake in...vivo of t r i t i a t e d thymidine into DNA occurred

a f t e r 3 hours exposure to the radioactive precursor due to degradati

Because of these considerations, another series o f

14
experiments was c a r r i e d out i n which the rats were given C-

thymidine 3 1/2 hours a f t e r r e c e i v i n g H - l a b e l l e d thymidine.

3

The animals were s a c r i f i c e d 5 or 10 minutes a f t e r the second

injection. The f r a c t i o n s of mucosal DNA from the MAK column

were assayed f o r r a d i o a c t i v i t y and u l t r a v i b l e t absorbance as

before. The data from these experiments are presented i n

Figures 8•to 11. The.elution p r o f i l e s of the DNA i s o l a t e d i n

the 5 minute experiment, as shown i n Figure 8," and 10 minute

 to
1 20 —1— -f-
HO so t,o- 70 90
80

Fig.9. Chromatography of mucosal DNA, l a b e l l e d with »H? thynSdine f o r 3 i hours and C - thymidine f o r 10
H

minutes, on MAK column.

 H 5 min.'c i n c o r p o r a t i o n

A.1Qmin. G i n c o r p o r a t i o n .
e

$5-

50! — n
'— — — r°-

ZS) j-— ± j-— —i —f :

f- i : i 1 1
' 2, 3 4 5 b 7 8 4 ^ I I

3 Fraction No.
F i g . ID. Ratio of r a d i o a c t i v i t y due to H/ o p t i c a l density of the DNA fraations obtained from MAK chromatography
• i n experiments i n which H- thymidine was administered to rats 3g- hours before i n j e c t i o n of C-thymidine.
Each fraction c o m p r i s e s one -tube-. F r a c t i o n no. 1 i s the f i r s t tube Q f t h e DNA peak.
 A 5 min. C incorporation

©10 m i n t incorporation

-4- H 1— 1—
z 3 H 5- 6 8 id

Fraction No.
3 /IA

F i g . 11, Ratio of H /^ C i n f r a c t i o n s comprising the DNA pe»k eluted by chromatography of DNA which was

l a b e l l e d with H- .thymidine f o r 3g- hours. /

 52

experiment, as shown i n F i g u r e 9, a r e s i m i l a r to that obtained

in e a r l i e r experiments i n t h e p r e s e n t works.

As i l l u s t r a t e d i n Figure 11, the d i s t r i b u t i o n of the

ratio of H/ 3 1 4
C among t h e DNA fractions followed a similar

pattern i n the 5 minute and 10 m i n u t e experiments. This ratio

reached a maximum a t f r a c t i o n 4 o r 5, a r e s u l t which was also

obtained i n the experiments i n which rats were e x p o s e d to

tritiated-thymidine f o r 24 h o u r s . As -.shown, i n . F i g u r e 10, the

distribution o f t h e r a t i o o f ^H/O.D. among t h e DNA fractions

was c o n s t a n t i n t h e 10 m i n u t e experiments. I t s seems therefore

3 14

that this distribution of the H/ C ratios is-characteristic

of these experiments i n which a n i m a l s were k i l l e d 5 o r 10

minutes after administration o f the second p r e c u r s o r . A t 20

3 14
minutes, the r a t i o of H/ C increases with f r a c t i o n number
3 14

a n d a t 40 m i n u t e s , the H/ C ratio i s constant throughout

the fractions (14) .

It i s possible that these r e s u l t s are i n d i c a t i v e of a

metabolic heterogeneity within t h e DNA m o l e c u l e s , as c o u l d be

expected from r e c e n t theories o f chromosome r e p l i c a t i o n (67,

72, 73). S m a l l m o l e c u l e s o f newly s y n t h e s i z e d DNA would

14
c o n t a i n t h e g r e a t e s t amount o f " C r e l a t i v e t o t h e amount o f
o l d , o r ^ H - l a b e l l e d DNA. As small molecules are eluted a t the.

b e g i n n i n g o f t h e DNA peak on MAK chromatography (£42,, 53."•'•)• the

3 14

H/ C ratio of the early.fractions s h o u l d be l o w - a n

observation found i n this experiment. After 5 o r 10 m i n u t e s ,

some o f t h e s e s m a l l m o l e c u l e s may h a v e f o r m e d loose aggregates

 53

(67, 73) w h i c h a r e h e l d t o g e t h e r b y weak h y d r o g e n bonds a n d

c o n t a i n l a r g e r e g i o n s o f d e n a t u r a t i o n as p o s t u l a t e d by

Salganik e t a l . (71). These molecules which are denatured

to v a r i o u s degrees will t e n d t o be e l u t e d last according to

the r e s u l t s o f Sueoka and Cheng (47) . S i n c e such molecules

are aggregates of the smaller metabolically active molecules

14 3 14

and h e n c e h a v e a h i g h p r o p o r t i o n o f C, t h e H/ C ratio

will begin to decrease i n the l a t e r fractions. Between t h e

s m a l l newly s y n t h e s i z e d m o l e c u l e s and t h e i r partially-denatured

aggregates i s t h e b u l k o f t h e DNA, w h i c h i s metabolically

stable. T h i s h y p o t h e s i s based on Sueoka's suggestion could

14

e x p l a i n the curves obtained i n those experiments i n which . C

thymidine incorporation into DNA was p e r m i t t e d f o r 5 o r 10

minutes.
A f t e r a 20 m i n u t e i n t e r v a l o f DNA m e t a b o l i s m i n the
14

presence of C-thymidine, some o f t h e s e s m a l l o r l o o s e l y

aggregated DNA m o l e c u l e s c a n be e x p e c t e d t o be i n c o r p o r a t e d

i n t o t h e m a t u r e DNA fraction ( 6 7 , 7 3 ) . Thus some o f t h e H - 3

14
l a b e l l e d DNA w i l l b e l a b e l l e d w i t h C, but. t h i s w i l l be a
m i n o r f r a c t i o n c o m p a r e d t o t h e t o t a l ' DNA w h i c h i s l a b e l l e d
3 3 14
with H. T h e n e t r e s u l t s h o u l d be an i n c r e a s i n g H/ C

ratio with f r a c t i o n a c c o r d i n g t o i n c r e a s i n g m o l e c u l a r s i z e and-

metabolic-stability o f t h e DNA fractions eluted from the :

column.

14
After 40 m i n u t e s , very l i t t l e C-thymidine w o u l d be

available f o r incorporation, and, a s a r e s u l t : , the incorporation

14
of the C-thymidine i n t o t h e newly formed material would
 54

decrease. A f t e r '40 m i n u t e s , t h e r e f o r e , t h e DNA i s stabilized

w i t h respectAasotope i n c o r p o r a t i o n . The b u l k o f t h e newly

synthesized molecules will be u n l a b e l l e d and t h e r e f o r e their

incorporation into the bulk o f t h e DNA w i l l h a v e no e f f e c t

3 14

on the isotope r a t i o . The p l o t of H/ C v e r s u s DNA fraction

w o u l d be e x p e c t e d to give a straight horizontal line.

Any c o n c l u s i o n s deduced from t h e above r e s u l t s must be

t e m p e r e d by c o n s i d e r a t i o n o f o t h e r f a c t o r s . F o r example,

cells i n the krypts of Lieberkuhn d i v i d e most r a p i d l y and

migrate to the tips of the v i l l i , where t h e y no l o n g e r divide

and are e v e n t u a l l y sloughed off. T h i s might introduce

variability of results from one e x p e r i m e n t t o another. It is

very probably t h a t the mucosal t i s s u e isolated from t h e

-small i n t e s t i n e i s contaminated w i t h muscle and b l o o d cells

as w e l l as b y s e c r e t i o n s f r o m glands and o r g a n s t h a t serve

the small intestine.

It i s n o t known w h e t h e r o r n o t a l l t h e m a t u r e DNA i s
3 14
l a b e l l e d with H and t h e n e w l y s y n t h e s i z e d m o l e c u l e s with C
3

and H o r whether the l a b e l l i n g i s incomplete. The r a t i o o f

3
H/O.D. i n t h e 3 1/2 h o u r e x p e r i m e n t indicates only a broadly,

uniform labelling pattern. S i n c e MAK chromatography depends

on three f a c t o r s working together, i t i s d i f f i c u l t t o know

what e f f e c t m o l e c u l a r size alone o f t h e DNA m o l e c u l e h a s on

its overall elution. A factor i n t h e 40 m i n u t e and p o s s i b l y

in t h e 20 m i n u t e e x p e r i m e n t s i s the rate of turnover of the

mature DNA.
 ndenatured

o native

F i g . 12. R^tio of ' H/

3
C i i i f r a c t i o n s comprising the DNA perk eluted by chromatography of DNA which w?s l a b e l l e d
i h

with H f o r 2U hours and '"C f o r 20 minutes. The comparison i s made between f r a c t i o n s that are denatured
3

by heating following chromatography before.radioactive counting and those-which had no treatment p r i o r

to counting.
 56

An i n t e r e s t i n g o b s e r v a t i o n was f o u n d i n counting the

3 14

DNA f r a c t i o n s . The d i s t r i b u t i o n of the H/ C ratio among t h e

fractions d e p e n d e d on t h e t r e a t m e n t o f t h e samples b e f o r e radio-

active counting. I n some o f t h e e x p e r i m e n t s t h e DNA samples

upon w h i c h T ' d e t e r m i n a t i o n s were made were t h e n used f o r

radioactive assay. As a r e s u l t , t h e DNA was h e a t denatured

and cooled before being processed f o rcounting. I n t h e case

of a 20 m i n u t e e x p e r i m e n t carried o u t under these circumstances,

3 14

the p a t t e r n o f t h e H/ C r a t i o was a s t r a i g h t line instead

of an i n c r e a s i n g curve typically observed. These r e s u l t s a r e

shown i n F i g u r e 12.

In order t o account f o r these differences, the steps

leading t o t h e c o u n t i n g o f t h e DNA f r a c t i o n s were e x a m i n e d i n

more d e t a i l as f o l l o w s . The DNA f r a c t i o n s from MAK .

c h r o m a t o g r a p h y were d i v i d e d into two e q u a l p o r t i o n s . One

portion o f each f r a c t i o n was d i a l y z e d separately against

distilled water and d u p l i c a t e samples o f each dialyzed

f r a c t i o n were a n a l y z e d f o rradioactivity. E a c h d i a l y s a t e was

evaporated t o s m a l l volume i n a f l a s h ^evaporator and t h e

optical d e n s i t y a t 260 nm a n d r a d i o a c t i v i t y o f each dialysate

were m e a s u r e d . T h e o t h e r p o r t i o n o f e a c h f r a c t i o n was

dialyzed against standard saline citrate. When t h e s e dialysates

were e v a p o r a t e d , the resulting high s a l t c o n c e n t r a t i o n made

counting impractical. The t h e r m a l denaturation temperature

of t h e DNA was d e t e r m i n e d i n each f r a c t i o n and t h e r e s u l t i n g

s o l u t i o n w h i c h now c o n t a i n e d d e n a t u r e d DNA was dialyzed

against d i s t i l l e d w a t e r and t h e u l t r a v i o l e t absorbance a t 260 nm

 57

and t h e r a d i o a c t i v i t y were measured. The d i s t i l l e d water

dialysate was e v a p o r a t e d t o s m a l l volume and a s s a y e d f o r

radioactivity and a b s o r b a n c y . Samples o f u n f r a c t i o n a t e d DNA

were s i m i l a r l y treated. The r e s u l t s from t h i s experiment are

p r e s e n t e d i n T a b l e s I I I , I V and V.

UV
14 . 14 3
Treatment O.D. H c
V c
1 4
O.D. H c
H/ C : Spectrum
3 3 14

no t r e a t m e n t 16 139,665 13,289 10.5 no d i a l j s a t e

dialyzed against 15 130,735 13^152 10.4 .770 - - - No X max a

d i s t i l l e d water 260 nm

T„ & dialyzed .881 8422.2 645 13.10 1.478 1675 79.1 21.1 X max a t
against d i s t . 260 nm
water o f a
s o l u t i o n con-
t a i n i n g 1.3
O.D. units.

TABLE I I I

E F F E C T OF VARIOUS TREATMENTS ON THE RADIOACTIVITY AND

ABSORBANCE OF A SOLUTION OF DOUBLY-LABELLED RAT

I N T E S T I N E DNA IN SALINE C I T R A T E .
 DNA S o l u t i o n Distilled Water Dialysate

Fraction Total
No. O.D. 3
H 14
c 3
H/ 1 4
C O.D. 3
H 1 4
c 3
H/ 1 4
C UV Spectrum
1
1 1.910 76.8 65 1.16 1.521 - - No X max a t 260 nm

2 2.960 9.41 32 .18 1.197 - - -

3 2.711 2396 509 4.71 0.823 - *

4 1.845 4359 445 9.79 0.907 - • - -

5 2.275 4638 484 9.57 1.007 - - - JX

6 1.917 4274 320 13.34 0.967 -

7 1.594 4185 185 22.65 0.672 - - - it

8 2.678 4144 220 18.81 0.786 - -

CO
9 1.080 4074 112 36.30 - - - . »
1.287

10 1.222 4062 92 • 44.39 1.108 - - - *

11 1.158 4035 56 71.67 0.993 - - -

12 0.660 4038 59 68.09 0.937 - - - JL

TABLE I V RADIOACTIVITY AND ABSORBANCY AFTER D I A L Y S I S AGAINST D I S T I L L E D WATER

OF FRACTIONS OF DOUBLY-LABELLED DNA OBTAINED BY CHROMATOGRAPHY ON MAK.

 DNA S o l u t i o n D i s t i l l e d Water Dialysate

Traction Total
T
No. O.D. 3
H
1 4
c H/ C M
O.D. 3
H
1 4
c 3
H/ 1 4
C UV Spectrum

1 1.552 49.2 - - 1.958 196 4 55.0 X max a t 260 nm

2 1.502 - 56.7 - - 2.210 214 41 5.0 II

H
3 1.338 786 151 5.2 89.2 0.956 224 42 5.36
II
4 1.678 4192 415 10.0 88.3 1.040 920 80 11.51
II
5 1.317 3624 377 9.6 87.4 1.297 194 34 3.74.
II
6 1.165 3030 307 9.8 86.8 1.010 305 16 18.73
II
7 1.513 990 112 8.45 86.2 1.192 233 16 14.15
II
8 0.927 651 105 6.2 85.6 0.920 839 57 14.79

9 1.015 767 81 9.5 85.6 1.412 453 52 8.65 II ui

10 1.024 615 64 9.5 - 1.349 647 51 12.69 It

11 1.065 291 70 4.1 - 1.606 553 •30 18.48 II

12 1.826 367 68 5.3 - 0.617 292 36 8.19 •I

T A B L E V RADIOACTIVITY AND ABSORBANCY AFTER HEATING AND THEN D I A L Y S I S AGAINST

D I S T I L L E D WATER OF FRACTIONS OF DOUBLY-LABELLED DNA OBTAINED BY CHROMATOGRAPHY ON MAK.

 bU

The data i n Tables IV and V i n d i c a t e that t h e amount o f

ultraviolet absorbing material i n t h e DNA s o l u t i o n w h i c h h a s

been d e n a t u r e d by h e a t i n g p r i o r to counting i s l e s s than that

of t h e DNA s o l u t i o n s not treated t h i s way. T h e r e is a simult-

aneous i n c r e a s e i n t h e amount o f u l t r a v i o l e t a b s o r b i n g material

recovered i n the d i s t i l l e d water d i a l y s a t e s o f the denatured

DNA s o l u t i o n s . T h e uv s p e c t r u m o f t h e d i a l y s a t e s from t h e

n a t i v e .DNA s o l u t i o n s shows no maximum a r o u n d 260 nm and h e n c e

the o p t i c a l density measured i s probably due t o t h e m a t e r i a l

1

released from t h e d i a l y s i s t u b i n g . On t h e o t h e r h a n d , t h e uv

spectrum o f t h e d i s t i l l e d water d i a l y s a t e s o f the denatured

DNA s o l u t i o n s show an a b s o r p t i o n maximum a t 260 nm, w h i c h

indicates the presence of nucleotide material i n the dialysates.

The r a d i o a c t i v i t y of the dialysates o f t h e d e n a t u r e d DNA solutions

also indicates that denaturation of nucleic acids led to

release o f small molecular weight p a r t i c l e s . •'

In the experiment i n w h i c h t h e DNA s o l u t i o n s were

denatured by h e a t i n g p r i o r t o d i a l y s i s a g a i n s t distilled water-,

the 3
H / 14c i s r e l a t i v e l y constant throughout the fractions.

When t h e DNA s o l u t i o n s received no t r e a t m e n t p r i o r t o d i a l y s i s

against distilled water, the ^H/ 14 c increased with increasing

fraction number.

In t h e d i a l y s a t e s o f t h e d e n a t u r e d DNA s o l u t i o n s , t h e

^H/ r a t i o was h i g h e r t h a n i n t h e c o r r e s p o n d i n g DNA solutions

remaining inside the d i a l y s i s tubing. This could be a r e s u l t o f

 61

3
preferential loss of H-labelled material through the d i a l y s i s
14
tubing or retention of C-labelled material inside the tubing.

An aliquot o f t h e DMA s o l u t i o n which contained approximately

16.0 O.D. u n i t s was c o u n t e d w i t h o u t a n y p r i o r treatment. As

shown i n T a b l e I I I , t h e u n f r a c t i o n a t e d m a t e r i a l as i s o l a t e d had
3 14 3 14
high counts o f both H and C w i t h a H/ C ratio o f 10.5..

Dialysis against distilled water ZLad.: t o o n l y a s l i g h t decrease

in the o p t i c a l density and r a d i o a c t i v i t y o f t h e DNA solution;

T h e r e was some uv a b s o r b i n g m a t e r i a l i n the d i a l y s a t e b u t no

radioactivity c o u l d be d e t e c t e d . The u v s p e c t r u m of the

d i a l y s a t e h a d no maximum a r o u n d '260 nm.; On t h e o t h e r h a n d ,

the distilled water d i a l y s a t e o f t h e h e a t d e n a t u r e d DNA sample

contained both r a d i o a c t i v i t y and uv a b s o r b i n g m a t e r i a l , with

3 14
•an - a b s o r p t i o n maximum a t 260 nm. The H/ C ratio o f the
d i a l y s a t e was 21.1 w h i c h s u g g e s t s t h a t more H - l a b e l l e d
3
than
14 .
C - l a b e l l e d m a t e r i a l passed through the d i a l y s i s tubing. The

optical density o f t h e d e n a t u r e d DNA solution before dialysis

was 1.130, w h i l e a f t e r dialysis t h e O.D. was 0.881. These

observations indicate that heat denaturation o f the doubly-

labelled DNA r e l e a s e d n u c l e o t i d e m a t e r i a l of small molecular

weight which passed through the d i a l y s i s tubing. S u t t o n and .

Petersen (85) o b s e r v e d that prolonged d i a l y s i s of the products

of t h e d e g r a d a t i o n o f DNA b y d i p h e n y l a m i n e i n formic acid

causes shorter oligonucleotides t o pass through the d i a l y s i s

tubing.
61 (a)
. 1 3 . Chromatography on DEAE - C e l l u l o s e of the d i s t i l l e d water d i a l y s a t e

of the mucosal DNA that h?d been denatured by heating p r i o r t o

d i a l y s i s . E l u t i o n was c a r r i e d out,with a .gradient of NaCl i n 7M urea-

T r i s H C l - pH 7.8
 63

An e x p e r i m e n t .was done t o t r y t o i d e n t i f y the m a t e r i a l •

which i s lost'-.on d i a l y s i s . A sample o f u n l a b e l l e d mucosal

DNA which contained a t o t a l o f 532 O.D. u n i t s was heated to

boiling and allowed to cool slowly. After dialysis against

.distilled water, t h e s o l u t i o n was found to contain a total of

398 O.D. u n i t s w h i l e i t s volume r e m a i n e d t h e same. The

dialysate c o n t a i n e d 114 O.D. units. The dialysate was

fractionated by a chromatographic procedure described by Lee

(86) w h i c h i s a modification o f t h e method o f T o m l i n s o n and Tener

(87) .

DEAE-cellulose (Whatman DE22) was prepared i n the

chloride f o r m as i n s t r u c t e d by the manufacturer, A 2 x 90 cm

column..was p a c k e d w i t h t h e e x c h a n g e r under a pressure of 5 p.s.i.

and equilibrated with 7 M urea c o n t a i n i n g 30 m l o f 0.1 IM T r i s -

HC1, pH 7.8 per l i t e r . The s a m p l e was loaded c a r e f u l l y onto

the t o p . o f t h e c o l u m n and e l u t i o n was carried out w i t h a '. v

gradient of sodium c h l o r i d e w h i c h was formed by two liters

of b u f f e r e d 7 M u r e a i n t h e m i x i n g chamber and two liters of

b u f f e r e d 7."M u r e a - 0 .I'M NaCl i n the r e s e r v o i r . Five ml

f r a c t i o n s .were c o l l e c t e d and e a c h f r a c t i o n was assayed for-

absorbancy a t 260 nm and sodium chloride concentration.

As shown i n F i g u r e 13, f o u r peaks of ultraviolet

a b s o r b i n g m a t e r i a l ' w e r e o b t a i n e d from the chromatographic

fractionation of the d i a l y s a t e . T h e y were e l u t e d w i t h salt

concentrations which are t y p i c a l f o r short chain nucleotide

material.. The peaks were n o t f u r t h e r analyzed f o r the

following reasons. In order to determine t h e number of

 64

molecules o f each chain length present, i t i s necessary to

know two o f t h e f o l l o w i n g f a c t o r s ; c h a i n length of the

oligonucleotide, total number o f m o l e s . o f b a s e Cor nucleotide)

present, or total amount o f p h o s p h a t e c o v a l e n t l y bound t o t h e

nucleotide material, provided t h e number o f m o l e s o f

phosphate (mole o f b a s e ) p r e s e n t i n each o l i g o n u c l e o t i d e i s

known, . Phosphate a n a l y s i s would i n d i c a t e t h e amount o f

"organic" phosphate i . e . phosphate i n t h e n u c l e o t i d e material,

present. However, i t i s n o t known how many m o l e s o f p h o s p h a t e

are p r e s e n t a t the terminal ends o f each o l i g o n u c l e o t i d e . The

c h a i n l e n g t h o f e a c h o l i g o n u c l e o t i d e c a n o n l y be s u r m i s e d

from i t s p o s i t i o n o f e l u t i o n r e l a t i v e to that of oligonucleotides

o f known c h a i n length i n t h e same c h r o m a t o g r a p h i c procedure.

Hydrolysis of the nucleotide material followed by paper

chromatography and q u a n t i t a t i v e s p e c t r o p h o t o m e t r y o f t h e s p o t s

would i n d i c a t e t h e t o t a l moles o f each base p r e s e n t . These

experiments a r e beyond t h e scope o f t h i s thesis.

DEGRADATION OF THE ISOLATED DNA BY SNAKE VENOM PHOSPHODIESTERASE

An observation from t h e d i a l y s i s e x p e r i m e n t s was that

the r a t i o o f H/ C was h i g h e r i n the d i a l y s a t e than i n the

d e n a t u r e d DNA solution. Heating w o u l d d e s t r o y -hydrogen b o n d i n g

w h i c h m i g h t be i n v o l v e d i n h o l d i n g small molecules together

to form aggregates as w e l l as t h o s e binding the. two c h a i n s o f

t h e DNA d o u b l e h e l i x together. A n u n b o n d e d e n d on one o f t h e

c h a i n s might be s u s c e p t i b l e t o c l e a v a g e during denaturation.

Terminal i n c o r p o r a t i o n o f one o r two n u c l e o t i d e s could produce

 65

t h e s e f r e e ends (24) . As terminal incorporation occurs at the

3'OH end (24). and s n a k e venom p h o s p h o d i e s t e r a s e cleaves from

the 3 ?
and y i e l d i n g 5' nucleotides, i t was decided to examine

the products of s n a k e venom p h o s p h o d i e s t e r a s e d e g r a d a t i o n for

radioactivity. The presence of a higher proportion of 3

H
14
relative to C i n the d i a l y s a t e m i g h t be a r e s u l t of terminal
3 •

incorporation of H-labelled precursor, w h i c h i f unbonded,

w o u l d be released from the DNA chain on heating and pass

through the dialysis tubing i n t o the dialysate. These enzyme

studies were c a r r i e d o u t on samples of doubly-labelled DNA

3
isolated f r o m r a t s w h i c h were e x p o s e d t o H-thymidine for 24
14

h o u r s and C-thymidine for 5 minutes. The following DNA

s o l u t i o n s were u s e d f o r these studies: a) a saline-citrate

-solution of the isolated, unfractionated DNA which contained

15.7 O.D. units b) the freeze-dried residue, containing 14.4

O.D. units of the e n t i r e peak o f DNA material obtained by

chromatography of the i s o l a t e d DNA on MAK c) the freeze-

dried residue, containing 12.5 O.D. units, of the entire peak

o f DNA material, o b t a i n e d by chromatography o f an aliquot of

the i s o l a t e d DNA on MAK followed by heat denaturation before

being freeze-dried. Each of t h e s e DNA s a m p l e s was dissolved

in 4.5 ml of 0.01M magnesium c h l o r i d e i n 0.04 M Tris buffer,

pH 8.9. One-half ml. of a s n a k e venom phosphodiesterase

solution, a gift of Dr. W. R a z z e l l , was a d d e d and the mixture

was incubated at 37°C. One ml. of the s o l u t i o n was removed

i m m e d i a t e l y and placed i n a micro-curvette. The increase in

optical density of t h i s sample was recorded automatically in ",

a Gilford automatic recording spectrophotometer with the cell
compartment maintained at 37°C. The remaining mixture was

incubated at 37°C and at various time intervals, 0.5 ml

portions of the m i x t u r e were removed and p l a c e d i n cold 12 ml.

Servall centrifuge tubes. Ten m i c r o l i t e r s of 1% s e r u m albumin

and 0.5 ml of ice-cold 20% trichloroacetic acid solution were

added t o each tube to stop the reaction. The resulting

suspension was centrifuged for 20 minutes at 30,000 g. The

acid-soluble fraction was removed w i t h a Pasteur pipette and

the acid-insoluble fractions were washed twice in 0.5 ml. of

1% t r i c h l o r o a c e t i c acid solution followed by centrifugation.

The washings were added t o the corresponding acid soluble

fractions'. The precipitates were then washed w i t h 2 ml. of

ethanol-ether mixture (1:1 v/v) and 2 m l o f anhydrous ether.

They were then d i s s o l v e d i n 1.0 ml of 0 . 1 M ammonium acetate

pH 6.5 - 0.01M M g C l . 2 Each s o l u t i o n was then assayed for

radioactivity.

Each a c i d - s o l u b l e fraction was extracted three times

with 5 ml of ether to remove the trichloroacetic acid and

the r e s i d u a l e t h e r w a s removed by b u b b l i n g n i t r o g e n through

the solution. The solutions were assayed for optical density

at 260 nm a n d r a d i o a c t i v i t y . The results of this experiment

are shown i n F i g u r e 14.

One observation from these graphs is that the rate of

release of nucleotides is greater i n t h e d e n a t u r e d DNA sample

than i n the o t h e r two samples.

 67,

The r e s u l t s obtained f o r the fractionated native DNA

sample and the unfractionated sample are very .similar and

d i f f e r markedly from those of the denatured fractionated sample

The rate of r e l e a s e of uv absorbing material i n t o the acid

soluble f r a c t i o n recorded automatically agreed with that

obtained by measuring the O.D. of the acid-soluble portions of

the a l i q u o t s w h i c h were r e m o v e d a t s p e c i f i c i n t e r v a l s .

3 14
The rate of r e l e a s e of H-labelled nucleotides and C-

labelled nucleotides i n t o the acid soluble fractions was

calculated from the r a d i o a c t i v i t y of the acid soluble portion

of each a l i q u o t . From t h e denatured, f r a c t i o n a t e d DNA sample,

3
the rate of r e l e a s e of the H-labelled nucleotides was greater
- 14

than that of- t h e C-labelled nucleotides . Since the rate

curves are s i m i l a r , there i s no indication of a marked

release o f one
w i t h r e s p e c t t o t h e o t h e r and t h e r e f o r e i t was
3 14
concluded that H and C are u n i f o r m l y d i s t r i b u t e d throughout.
the c h a i n . From t h e f r a c t i o n a t e d ' n a t i v e and unfractionated
3

DNA s a m p l e s , t h e release of H-labelled material occurs at

a low level corresponding to a slow r e l e a s e of acid soluble

material.. This observation suggests, t h a t the 3

H-labelled

material o c c u r s w e l l w i t h i n t h e DNA s t r a n d s . On t h e o t h e r
14
hand, the r e l e a s e o f C - l a b e l l e d n u c l e o t i d e s i n t o the a c i d
s o l u b l e f r a c t i o n , e s p e c i a l l y from the u n f r a c t i o n a t e d DNA,
occurs slowly at first and a f t e r 20 minutes there is a
14

release of C followed by a tapering off. This is the

expected r e s u l t i f t e r m i n a l incorporation of a labelled

precursor has occurred.

 Time in minutes
Fig. 14. Release of u l t r a v i o l e t absorbing material ( • ) and r a d i o a c t i v i t y -
3 H (O ) and C (• ) - into the acid soluble f r a c t i o n upon digestic
,M

of doubly-labelled mucosal DNA with snake venom phosphodiesterase.

The^E/^C r a t i o i n the acid-insoluble f r a c t i o n (A) i s also sho'/n.
 68

The distribution of the labelled nucleotides throughout

3 14

the DNA chain can a l s o be i n d i c a t e d by the H/ C ratio of the

acid-insoluble fraction. I f i t remains constant, as in the

case of the d e n a t u r e d , f r a c t i o n a t e d DNA sample, the two labelled

nucleotides are probably evenly d i s t r i b u t e d throughout the

chain. On the other hand, a sharp i n c r e a s e or decrease i n this

ratio could i n d i c a t e a m a r k e d l o s s o f one of the nucleotides

with respect to the other. Concurrent with the observed
14
release of C-labelled acid soluble material i s an increase
3 14

in the H/ C ratio of the a c i d - i n s o l u b l e f r a c t i o n s of the native

f r a c t i o n a t e d and u n f r a c t i o n a t e d DNA samples.

The r e s u l t s o b t a i n e d by C. M e z e i (14) f o r DNA exposed
14

for 20 or 40 minutes to C-thymidine are s i m i l a r to those with

the denatured 5 m i n u t e DNA. On the other hand, the data from

the native 5 m i n u t e DNA s u g g e s t t h a t t h e r e i s a t e r m i n a l

14
i n c o r p o r a t i o n of C - l a b e l l e d thymidine n u c l e o t i d e . A possible
e x p l a n a t i o n f o r t h e s e d i f f e r e n c e s may be t h e f o l l o w i n g . After
3
t h e DNA m o l e c u l e s have been r e p l i c a t i n g i n the presence of H-
3
thymidine f o r 24 hours, the H may become u n i f o r m l y d i s t r i b u t e d
throughout the chains. During the 20 minute pr 40 minute
. 14

interval i n which C-thymidine i s a precursor f o r DNA, the small,

newly s y n t h e s i z e d DNA molecules (highly l a b e l l e d with 1 4

C),

w i l l become i n c o r p o r a t e d i n t o the m a i n DNA chains and so the

net result will be a group of fairly uniformly labelled DNA

molecules-. T h e r e may be some t e r m i n a l i n c o r p o r a t i o n of the
14
C-labelled nucleotide, but this may be m a s k e d by a greater
14
proportion of C-labelled material well within the DNA chain as
 69

a result o f DNA replication.

During a short interval of exposure to 1 4

C-thymidine,

the degree of DNA replication will not be as great and any

terminal i n c o r p o r a t i o n of 1 4
C - l a b e l l e d nucleotides might be

clearly i n d i c a t e d as. i n t h e case of the native unfractionated

and f r a c t i o n a t e ' ! DNA s a m p l e s . The r e s u l t s of the denatured

fractionate-.?.' DNA sample s u g g e s t that-fche terminally incorporated

^"^C-thymidine i s l o s t during denaturation. However, t h i s observation

has not been r e p o r t e d by other workers.

The above r e s u l t s , h o w e v e r , do not e x p l a i n the presence

of l a r g e amounts o f 3
H-labelled material i n the dialysates

a f t e r d e n a t u r e d DNA material i s d i a l y z e d . I t may be that the

3
H-labelled material occurs to a great degree a t the 5' end.

An experiment which could indicate this would i n v o l v e degradation

of the DNA by phosphodiesterase I I , which cleaves from the 5"

end.

The Purification and Counting of the Interphase Layers of the

Tissue Extracts Formed D u r i n g the I s o l a t i o n of the Mucosal DNA

After the DNA containing aqueous l a y e r had been carefully

removed, the interphase l a y e r formed d u r i n g the deproteinization

with p h e n o l was purified and prepared for radioactive counting

as o u t l i n e d by C.Mezei ( 7 8 ) . The phenol l a y e r , which remained

in the c e n t r i f u g e tube a f t e r the aqueous l a y e r and the interphase

had b e e n r e m o v e d , was a l s o p u r i f i e d and a l i q u o t s of the

purified s o l u t i o n , were c o u n t e d ( 7 8 ) .

In d o u b l e - l a b e l l i n g experiments i n which 3
H-thymidine
14

was a d m i n i s t e r e d 24 h o u r s b e f o r e C-thymidine, some radio-

activity due t o H was o b s e r v e d i n b o t h t h e i n t e r p h a s e a n d

3

14
phenol l a y e r s . When t h e t i m e i n t e r v a l o f . C - i n c o r p o r a t i o n
14

was 5 m i n u t e s , the recorded C cpm were v e r y h i g h i n b o t h t h e

i n t e r p h a s e and t h e p h e n o l layers. This finding i s attributed

to the presence o f small molecular weight newly-synthesized

14
DNA m o l e c u l e s , h i g h l y labelled with C, m t h e i n t e r p h a s e and
14

C-thymidine, which had n o t y e t been i n c o r p o r a t e d i n t o the

DNA, i n the phenol layer. Attempts were made t o i s o l a t e t h e

DNA m a t e r i a l from t h e i n t e r p h a s e b u t these d i d n o t prove t o be

.successful.
I s o l a t i o n and C h a r a c t e r i z a t i o n o f t h e P y r i m i d i n e I s o s t i c h s

Other chromatographic t e c h n i q u e s were t r i e d i n an attempt

to f i n d a method f o r s e p a r a t i n g t h e DNA m o l e c u l e s a c c o r d i n g t o

o n l y one p a r a m e t e r i . e . molecular size, base composition or

degree o f hydrogen bonding. With this i n mind, b e h z o y l a t e d -

DEAE-cellulose, a g i f t f r o m D r . T e n e r , was u s e d to fractionate

t h e DNA samples. S e v e r a l peaks of u l t r a v i o l e t absorbing

m a t e r i a l were o b t a i n e d o n e l u t i o n ; but the observed fractionation

d i d n o t appear to. f o l l o w any p a r t i c u l a r pattern.

A slightly different p r o c e d u r e was u s e d as a p o s s i b l e

means o f e l u c i d a t i n g t h e c o m p l e x p r o c e s s o f DNA metabolism-.

 71 •

Cerny et a l . (40) reported the separation of pyrimidine isostichs

according to chain length and then base composition. As the

labelled precursor of DNA g i v e n to the animals is thymidine, a

pyrimidine nucleoside, i t was thought that if the. isostichs

could be identified, i t w o u l d be possible to know t o which

pyrimidines the labelled t h y m i d i n e was bound.

Pyrimidine isostichs are the fragments of the DNA c h a i n

which remain after the purine'bases have been removed by hydrolysis

with formic acid-diphenylamine. Their chain lengths are

determined by the number of pyrimidine bases existing in sequence

along the DNA c h a i n . Spencer et al.(88) have shown that

pyrimidine isostichs of different chain lengths can be separated

by chromatography on a D E A E - c e l l u l o s e column. Isostichs up to

10 nucleotides l o n g have been isolated by Cerny et al.(40) using

this procedure. They then subfractionated these isostichs of

identical chain length according to their base composition by

fractionation on a D E A E - c e l l u l o s e column at pH 3.1 (40). Thus,

they Were able to isolate pyrimidine isostichs of identical

chain length and base composition from'the heterogenous mixture.

Similar experiments were tried in this investigation i n which

DNA was isolated from rats w h i c h were allowed to incorporate

3 14
H-thymidine for 3 1/2 hours and C-thymidine for 5 or 10

minutes.

. T h e DNA w a s hydrolyzed according to the method of Burton

and P e t e r s e n (89). F o r t y m g . DNA a n d 25 ml. formic acid-

diphenylamine were incubated at 30°C for 18 hours i n the dark.

 72

The h y d r o l y s a t e was then diluted t o 200 ml. with distilled

w a t e r and cooled. The greenish-white p r e c i p i t a t e was filtered

u n d e r vacuum t h r o u g h a fine-grade sintered-glass f i l t e r and the

filter was washed w i t h 40 ml distilled w a t e r t o remove any

trapped oligonucleotides. The combined filtrate was evaporated

to 20 m l . on a flash evaporator. Another 200 ml distilled

w a t e r were a d d e d and the s o l u t i o n was re-evaporated. The

addition of d i s t i l l e d w a t e r and subsequence e v a p o r a t i o n was

repeated until t h e pH of the s o l u t i o n was 3.5. The diphenylamine

p r e c i p i t a t e was removed by filtration through a fine-grade

sintered-glass filter and washed w i t h 20 m l . distilled water.

The total filtrate was neutralized with 0.1N NH^OH and the

final v o l u m e was made up t o 80 m l with 0.01M lithium acetate

b u f f e r pH 5.3.

The D E A E - c e l l u l o s e used f o r chromatography o f the

h y d r o l y s a t e was obtained from Whatman (DE-32 M i c r o g r a n u l a r )

and sieved prior to use. Only the m a t e r i a l which passed through

a 200 mesh s i e v e was used. The D E A E - c e l l u l o s e was washed with

c y c l e s o f 0.5'N NaOH and 0.5IN/HC1 as recommended by the

manufacturer and following a final rinsing i n a b s o l u t e ethanol',

it was dried in air. E q u i l i b r a t i o n was a c h i e v e d by suspending

f i v e grams o f t h e powder i n 30 m l _ o f 0.1M acetic acid and

adjusting t h e pH t o 5.3 with l i t h i u m hydroxide. After the

s l u r r y had settled, the supernatant c o n t a i n i n g the fines was

decanted. One hundred ml. o f 0.01M lithium a c e t a t e pH 5.3 were

a d d e d and the s l u r r y was poured into a 25 cm x 1.5 cm column.

The c o l u m n was washed w i t h t h i s buffer until t h e pH and O.D. at

 73

260 run o f e f f l u e n t w e r e t h e same a s t h o s e of the eluent. The

neutralized dilute hydrolysate was a b s o r b e d on t h e column, which

was t h e n washed w i t h starting buffer until the effluent solution

was free of ultraviolet absorption. A salt gradient, formed by

1 liter o f 0.01 M l i t h i u m acetate buffer pH 5.3 i n t h e m i x i n g

chamber a n d a h e q u a l v o l u m e o f . O.0|.':.M l i t h i u m a c e t a t e pH 5.3-

O.HM L i C l pH 5.3, i n t h e r e s e r v o i r . , was u s e d for elution.

F i f t e e n ml. fractions were c o l l e c t e d a n d e a c h f r a c t i o n was

assayed for absorbancy a t 260 nm a n d conductivity.

The fractions within e a c h peak o b t a i n e d f r o m t h e above

c h r o m a t o g r a p h y were c o m b i n e d . The-pH o f t h e s o l u t i o n s was

adjusted t o 5.0 a n d t h e v o l u m e o f e a c h solution was m e a s u r e d .

The O.D-,,,. '} df e a c h solution was m e a s u r e d and t h e t o t a l number

2.70 HON.

of moles o f t h y m i d y l i c acid and c y t i d y l i c acid i n each solution

was calculated from the following equation, according to

Spencer e t al.(98)

zx-moles- b a s e = oAn** 7<>

" c * volume i n l i t e r s

O.D. " •' 27 0 x Volume (liters)

8.68 x 10 J

270
where ^ ^ for thymidylic acid and d e o x y c y t i d y l i c acid i s
3
8.68 x 10 . This equation c a n be u s e d for this calculation

since a t pH 5.0, b o t h thymidylic acid and d e o x y c y t i d y l i c acid

have'the', same-.;•' a b s o r b a n c e a s 270 nm.

 74

Inorganic and t o t a l phosphorus i n the i s o s t i c h fractions

were d e t e r m i n e d b y t h e method o f K i n g (91). I f the chain

length of the i s o s t i c h and o r g a n i c phosphate p r e s e n t a r e known,

the t o t a l moles o f base p r e s e n t i n each isostich f r a c t i o n can

be calculated from the formula Py ."p.. w h e r e Py refers to

n
J r
(n4=l) n
J

t h e number o f m o l e s o f p y r i m i d i n e p r e s e n t i n each isostich.

During the formic acid-diphenylamine hydrolysis, the purine

b a s e and s u g a r a r e removed leaving a p h o s p h a t e on e a c h e n d o f

the isostichs and hence t h e f o r m u l a f o r t h e number o f m o l e s o f

phosphate present i n each i s o s t i c h is(n>l]«

The radioactivity o f e a c h i s o s t i c h was a l s o measured.

The results a r e shown i n F i g u r e s 15 and 16 a n d T a b l e s V I a n d V I I .

Isostich Volume O.D. umoles H

3 1 4
c
No. (ml) 270/ml. pyrimidine cpm/ml. cmp/ml

I 26 0.172 520 40 6
II 56 0.113 736 112 96
III 97* 0.210 2328 2966 72
IV 111 0.260 3330 2112 74
V 55 0.127 770 3500 72
VI 70 0.258 2100 6828 94
VII 42 0.120 506 3820 74
VIII 88 0 .118 1014 4960 91

TABLE V I - RADIOACTIVITY AND -ABSORBANCY• * o f t h e p y r i m i d i n e isostichs

isolated by chromatography on D E A E - C e l l u l o s e of the

formic-acid-diphenylamine h y d r o l y s a t e o f mucosal DNA,

w h i c h was a l l o w e d to incorporate H 3
f o r 3 1/2 h o u r s a n d
14
C f o r5 minutes.
 L *

F i g . 15- Chromatography on DEAE - Cellulose of the diphenylamine - formic acid hydrolysate of DNA, which
3 ' 14

I had been l a b e l l e d with H f o r 3g- hours 'and C f o r 5 minutes. E l u t i o n was carried out with a
gradient of L i C l i n l i t h i u m acetate - pH 5.3 ' ' • '
 40
T u b e N o .
F i g . 16. Chromatography on DEAE - Cellulose of the diphenylamine - formic acid hydrolysate of DNA, which had
3 U
been l a b e l l e d with H f o r 3§- hours and C f° r
10-'aainutes. E l u t i o n was c a r r i e d out with a gradient

of L i C l i n l i t h i u m acetate - pH 5.3
 77

Isostich Volume O.D. umoles

3
H 1 4
c
No. (ml) 270/ml. Pyrimidine cpm/ml cpm/ml

I 42 2.153 10,500 172 48

II 41 0.851 4,018 512 96
III 174 0.740 14,964 850 108
IV 56 0.692 4,480 800 98
V 117 0.143 1,872 72410 2200
VI 90 1.510 15,570 4932 448

TABLE V I I - RADIOACTIVITY AND ABSORBANCY o f t h e P y r i m i d i n e I s o s t i c h s

isolated b y c h r o m a t o g r a p h y on D E A E - C e l l u l o s e of the

formic acid-diphenylamine h y d r o l y s a t e o f mucosal DNA,

w h i c h was allowed to incorporate H 3

f o r 3 1/2 h o u r s and
14
C f o r 10 minutes.

The elution profile f o r t h e 5 m i n . DNA, as i l l u s t r a t e d .

i n Figure 15, shows t h e p r e s e n c e of eight separate isostich

fractions. The number o f m i c r o m o l e s o f b a s e p r e s e n t i n each

i s o s t i c h was c a l c u l a t e d according t o the spectrophotometric

m e t h o d o f S p e n c e r e t al» ( 9 0 ) and t h e v a l u e s are l i s t e d i n Table

VI. The r a d i o a c t i v i t y due t o t r i t i u m i s located mainly i n

fractions VI and V I I I as i n d i c a t e d by t h e r a t i o o f H-cpm/no.

3

of ;'yumoles o f p y r i m i d i n e . On t h e o t h e r h a n d , t h e r a t i o o f
14
C cpm/no o f pinoles o f pyrimidine indicate that isostichs II
14
and VIII contained a greater proportion of C-^labelled m a t e r i a l .

In the experiment with t h e 10 m i n . DNA, only s i x peaks

were o b t a i n e d a s shown i n F i g u r e 1 6 . Approximately 90% o f t h e

3 14
Hand 75% o f t h e C were r e c o v e r e d m the f i f t h isostich.

In order t o s u b f r a c t i o n a t e these isostich fractions

accordings t o base composition, i t i s necessary to pool the

isostichs of i d e n t i c a l chain length which are obtained f r o m one

or two'other f r a c t i o n a t i o n s i n order t o o b t a i n enough m a t e r i a l "

 78

to be d e t e c t e d on elution. However i n e a c h labelling

experiment using 2 rats, approximately 30 mg o f DNA can be

isolated, a l l o f w h i c h i s r e q u i r e d f o r one formic acid-hydrolysis

and chromatography experiment. Hence t h e r e was not enough DNA

to repeat the fractionation. The tentative results obtained in

these i n v e s t i g a t i o n s , suggest that there are sequences of

homogeneous DNA molecules which have d i f f e r e n t metabolic

activities. These r e s u l t s suggest that these experiments should

be e x t e n d e d , i n w h i c h l a r g e amounts o f DNA are collected.

 79

SUMMARY

3 14

1. The p a t t e r n of incorporation i n vivo of H- and C-labelled

thymidine i n t o DNA o f r a t i n t e s t i n a l mucosa was u s e d as a

means o f e l u c i d a t i n g t h e c o m p l e x p r o c e s s o f DNA metabolism.

2. The l a b e l l e d DNA/ isolated f r o m t h e mucosa, was fractionated

on a column o f me'thylated-albumin k i e s e l g u h r , w h i c h separated

t y p e s o f DNA a c c o r d i n g to base composition. The G + C

c o n t e n t o f t h e DNA, ranged f r o m 48.1% i n t h e f i r s t fraction

to 37.0% i n t h e l a s t fraction o f t h e DNA material eluted

f r o m t h e MAK column.

3. The l a b e l l i n g e x p e r i m e n t s were a c o n t i n u a t i o n o f t h o s e

p r e v i o u s l y done i n t h i s laboratory. In the e a r l i e r

3
•experiments, H-thymidine was injected into the r a t s and
14

24 h o u r s later C-thymidine was administered. Twenty o r

f o r t y minutes after this second injection, t h e a n i m a l s were

sacrificed a n d t h e i n t e s t i n a l DNA i s o l a t e d . I n t h e 40 m i n .
3 14
experiment t h e H/ C r a t i o o f t h e DNA f r a c t i o n s f r o m MAK
chromatography was c o n s t a n t , b u t i n the- 20 m i n . experiment,
3 14

the H/ C ratio increased with increasing fraction number.

This observation suggested the p o s s i b i l i t y of metabolic

heterogeneity occurring within the mucosal DNA. In the

p r e s e n t i n v e s t i g a t i o n , t h e a n i m a l s were s a c r i f i c e d 5 m i n .
14
after injection of C-thymidine. The r e s u l t s s u b s t a n t i a t e
the e a r l i e r f i n d i n g o f m e t a b o l i c h e t e r o g e n e i t y i n t h e DNA.
3 14
In t h e 5 mm. experiment, the H/ C ratio increased to a

maximum a t f r a c t i o n 4 o r 5 a n d then' d e c r e a s e d . These

results i n conjunction with e a r l i e r f i n d i n g s were interpreted

 80

to indicate that newly synthesized, small molecular weight

14

DNA, w h i c h was highly labelled with C was eluted at the

beginning of the DNA p e a k f r o m MAK c h r o m a t o g r a p h y . The low

H/ C ratio in the later fractions of the 5 min. experiment

c o u l d be loose aggregates of these small molecules which

are held together by hydrogen bonds. As the interval of

14

DNA s y n t h e s i s i n the presence of C-thymidine increased,

two p r o c e s s e s occur. First, the n e w l y s y n t h e s i z e d DNA

m o l e c u l e s - g r a d u a l l y become incorporated into the high

m o l e c u l a r w e i g h t s t a b l e DNA f r a c t i o n , w h i c h i s l a b e l l e d w i t h
3 14
H. A f t e r 25 m i n u t e s , v e r y l i t t l e C-thymidine remains for
i n c o r p o r a t i o n i n t o DNA a n d t h e DNA m o l e c u l e s n e w l y s y n t h e s i z e d
3 14

are mostly unlabelled and t h u s after 40 m i n u t e s , the H/ C

ratio is constant throughout the fractions.

It was found that a f t e r a 24 hour incorporation with 3
H -
3
thymidine, the r a d i o a c t i v i t y due to H was not uniformly
distributed throughout the DNA f r a c t i o n s from the MAK c o l u m n .
3

This was shown b y the variation m the H to absorbancy ratio.

This finding is attributed to the high turnover rate of cells

in- the intestinal mucosa. Experiments were carried out

similar to those above except that the period of exposure
3
to H-thymidine was 3 .1/2 hours. Shortening the exposure
3 14

time to 3 1/2 hours d i d not alter the H/ C pattern in the

double incorporation experiments.

3 14

The observed pattern of the H/ C ratios was influenced by

the manner i n which the DNA w a s treated before counting. If

the 20 m i n . DNA f r a c t i o n s were denatured by heating, the

 81

3 14
H/ C r a t i o was constant throughout the fraction. If this
3 14

t r e a t m e n t was not c a r r i e d out, the pattern of H/ C ratio

was a rising curve, as found in earlier investigations.

Quantitative examination of the processes involved after

the i s o l a t i o n of the DNA indicated that small molecular weight

o l i g o n u c l e o t i d e m a t e r i a l was cleaved from the DNA sample

during denaturation and during the following dialysis step

against distilled water, t h i s nucleotide m a t e r i a l was lost

through the dialysis tubing.

3 14

The H/ C ratio of the nucleotide m a t e r i a l which passed out

of the s o l u t i o n of the d e n a t u r e d DNA sample i n t o the dialysate

was higher than t h a t of the d e n a t u r e d DNA. This finding

3

suggested a p r e f e r e n t i a l r e l e a s e of H-labelled material

into the d i a l y s a t e , which could have been incorporated

terminally i n t o the DNA molecules and released during heat

denaturation. However, e x p e r i m e n t s i n w h i c h t h e denatured

and n a t i v e DNA m o l e c u l e s were d e g r a d e d w i t h s n a k e venom

14

phosphodiesterase, indicated that the C-thymidine might be

incorporated t e r m i n a l l y i n a p r e f e r e n t i a l manner. Another

p o s s i b i l i t y which remains i s t h a t the preferential release

of 3
H-labelled material took p l a c e from the 5"* end. This

p o i n t was not tested.

Other chromatographic techniques were u t i l i z e d i n an attempt

to find a means o f separating DNA molecules according to

size only, since S u e o k a and Cheng had reported that MAK

chromatography allows the separation o f DNA molecules

according to three parameters. No conclusive r e s u l t s were

 82

obtained with fractionation of benzoylated DEAE-cellulose.

3 14

The pattern of H/ C ratio was irregular and this result

w o u l d n o t be expected i f c h r o m a t o g r a p h y was based on the

molecular size o f t h e DNA fractions.

8. DNA s a m p l e s were t r e a t e d ! w i t h formic acid-diphenylamine and the

pyrimidine i s o s t i c h s were i s o l a t e d . The 5 and 10 minute

s a m p l e s were a n a l y z e d by this procedure. On.the 5-minute

experiment, the radioactivity due to H3

was located mainly
14
in i s o s t i c h s . VI and VIII, while t h a t due to C was i n
isostichs I I and VI. In the 10 m i n u t e e x p e r i m e n t the radio-
3 14

activity due to both H and C was located i n isostich V.

While these f i n d i n g s may indicate t h a t i n c o r p o r a t i o n does

n o t .occur i n t o a l l pyrimidine clusters to the same extent,

. further confirmatory experiments s h o u l d be carried out.

 83

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