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University of Southern California Dissertations and Theses
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Studies of aminodeoxyuridine, methylaminodeoxyuridine and dimethylaminodeoxyuridine
(USC Thesis Other)
Studies of aminodeoxyuridine, methylaminodeoxyuridine and dimethylaminodeoxyuridine
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Content
STUDIES O P AMINODEOXYURIDINE,
M ETHYLAMINODEOXYURIDINE AND
DIMETHYL AMINODEOXYURIDINE
by
Susan P ran ces Kabat
b
A T h e sis P re sen te d to th e
FACULTY O P THE DEPARTM ENT O P BIOCHEMISTRY AND NUTRITION
UNIVERSITY O P SOUTHERN CALIFORNIA
In P a r t ia l F u lfillm e n t o f th e
R equirem ents fo r th e Degree
M aster o f S c ie n c e
January 1964
U M I Number: EP41352
All rights reserved
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UMI EP41352
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Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6
U N IV E R SIT Y O F S O U T H E R N C A L IF O R N IA
T H E GRADUATE SC H O O L
U NIVERSITY PARK
L O S A N G E L E S 7. C A LIFOR N IA
B ier 6 ^ KU
This thesis, written by
Susan Prance s.J a b a t
under the direction of h.&£..Thesis Committee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
M aster o f ..S cien ce
Dean
THESIS COMMITTEE
Chairman
TABLE OP CONTENTS
Page
LIST OP TABLES.................................... . ............................... i i i
LIST O P FIGURES ....................................... i v
LIST O P ABBREVIATIONS...................................................... . y
Chapter
I . INTRODUCTION .................................. I
I I . HISTORICAL BA C K G R O U N D .............................. 5
Growth, I n h ib it io n and In c o r p o r a tio n
S tu d ie s ,
E f f e c t s o f In co rp p ra ted A nalongs on
S tr u c tu r e and F u n ctio n o f D N A
S tu d ie s o f A m inouracil and R ela ted
D e r iv a tiv e s
I I I . EXPERIMENTAL ....................................... 15
Growth E xperim ents
S y n th e s is o f 2 - ^ C Ami no deoxy u r id in e
I s o l a t i o n , P u r if ic a t io n , and H y d ro ly sis
o f D N A
IV. RESULTS ........................................................... 24
Growth E xperim ents
A n a ly s is o f DNA H y d ro ly sa te
Y. DISCUSSION................................... 56
VI. SU M M A R Y . . . . . . . . . . .....................
BIBLIOGRAPHY .......................................................... 46
i i
LIST O F TABLES
T able Page
I . M axim al_V iable Count A tta in ed by E. c o l i
K12 T~ in th e P resen ce o f V ariou s A nalogs
and Minimal Thymidine . . . . . . . . . . 25
I I . M axim al_V iable Count A tta in e d by E. c o l i
K12 T” in th e P resen ce Of A nalogs and
Minimal Thymine . . . . . . . ................... 29
I I I . Maximal V ia b le Count A tta in ed by E. c o l i
15 T~ in th e Pr.esence o f A nalogs and
Minimal Thymidine . .. . . . . . . . . . . 32
IV. Maximal V ia b le Count A tta in ed by E. c o l i
15 T_ in th e P resen ce o f V arious Concen
t r a t io n s o f A T J D R . . . . . . . . . . . . 32
V. P u r if ic a t io n o f DRA from E. c o l i 15 T~ 33
V I. P ro p o rtio n o f B a ses i n E. c o l i , 15 T " * D J 5 T A . . 39
LIST O F FIGURES
FIGURE PAG E
1 . R e la tiv e Maximal V ia b le Count A tta in ed by
E. c o l i K12 T~ in th e P resen ce o f V arious
A nalogs and Minimal Thymidine . . . . . . 26
2 . Changes in T u rb id ity o f C u ltu res o f E . c o l l
K12 T” in th e P resen ce o f AUD R or M A U D R
and M inimal Thymidine . . . . . . . . . . 27
3 . Changes in V ia b le C e ll Count o f E. c o l l
K12 T \ during In cu b a tio n in th e P resen ce
o f AUDR or M A U D R and Minimal Thymidine . . 28
4 . Changes in V ia b le C e ll Count o f E. c o l i
15 T~ d u r in g .In cu b a tio n in th e P resen ce
o f AUDR or M A U D R and Minimal Thymidine . • 31
5 . G radient E lu tio n o f a H yd rolysate o f D N A
from .E . c o l l 15 T~ Grown in th e P resen ce
o f 1 C-AUDRand Minimal Thymidine . . . . 35
i v
LIST OF ABBREVIATIONS
O
A- angstrom u n it
AULR- aminode o x y u rid in e
AUR- am in ou rid in e
BU- 5-b rom ou raciI
BUDR- 5-brom odeoxyuridine
CltJ- 5- c h lo r o u r a c il
C1UDR- 5-c h io r o d e o x y u r id in e
dAMP- d eoxyad eon osin e monophosphate
dCMP- d e o x y e y tid in e monophosphate
dGMP- d eoxygu an id in e monophosphate
dUMP- d eo x y u rid in e monophosphate
D B ilA U D R - d i me t hylam inode o x y u rid in e
DNA- d e o x y r ib o n u c le ic a c id
DNAase- d eo x y rib o n u c lea se
FU- f lu o r o u r a c il
i u - io d o u r a c il
IUBR- io d o d e o x y u r id in e
M AUDR- m ethylam in odeoxyurid ine
RNA- r ib o n u c le ic a c id
RNAase- r ib o n u c le a s e
TDR- th ym id in e
TMP- th ym id in e monophosphate
UDR- d eo x y u rid in e
m olar e x t in c t io n c o e f f i c i e n t
m illim ic r o n mt\-
\ \ M m icrom oles
CHAPTER I
INTRODUCTION
The n a tu re o f th e s tr u c tu r e and fu n c tio n o f
d e o x y r ib o n u c le ic a c id (DNA) i s o f c o n s id e r a b le cu rren t
i n t e r e s t and im p o rta n ce. The normal ch em ica l co m p o sitio n
o f DNA from v a r io u s organ ism s i s known ( 1 ) . P h y s ic a l
s tr u c tu r e and th r e e d im en sio n a l c o n fig u r a tio n o f DNA h as
b een s tu d ie d by means o f X -ray c r y s ta llo g r a p h y and o th e r
p h y s ic a l m ethods ( 2 ) . In 1944- A very, MacLeod and McCarty
showed c l e a r l y th a t DNA c a r r ie s h e r e d ita r y in fo r m a tio n (3 ).
The work o f B eadle and Tatum on a u x o tro p h ic m utants o f
N eurospora c r a s s a le d t o th e "one g en e— one enzyme"
th e o r y , w hich i d e n t i f i e d one o f th e prim ary fu n c tio n s o f
D N A -—th e d e te r m in a tio n o f p r o t e in s tr u c tu r e (4-). R ecent
work h as su g g e ste d t h a t DNA d eterm in es a p r o te in s tr u c tu r e
by f i r s t b r in g in g about th e s y n th e s is o f a s p e c i f i c r ib o
n u c le ic a c id (RNA) m o le c u le , or m essenger (5 )* DNA i s
a ls o th ou gh t to have com plex r e g u la to r y fu n c tio n s i n th e
c e l l ( 6 ) . These are n ot y e t c o m p le te ly u n d ersto o d .
The s tr u c tu r e and. pathw ays o f b io s y n t h e s is o f th e
two p y rim id in e and two p u rin e b a se s w hich are th e p r in c i
p a l com ponents o f DNA are known ( 7 ) . Many a n a lo g s o f
th e s e fo u r normal com ponents have b een s y n th e s iz e d ( 13-17) ,
or found i n n a tu r e . Some, such a s 5 -b ro m o u ra cil (BU)
1 ____________________________________________
2
have been found to i n t e r f e r e w ith th e s y n th e s is and u t i l i
z a t io n o f normal DNA com ponents, presum ably by a c tin g a s
a c o m p e titiv e s u b s tr a te f o r enzymes in v o lv e d in de novo
s y n th e s is o f n u c le o t id e s , or fo r th e k in a s e s and polym er
a se in v o lv e d i n DNA s y n t h e s is from m u c le o tid e s ( 25- 2 7 ) .
In a d d it io n , s e v e r a l a n a lo g s have b een shown t o be
in c o r p o r a te d in t o DNA ( 2 8 - 3 0 ) .
C om p etition s t u d ie s have i n d i r e c t l y p ro v id ed
in fo r m a tio n about th e s p e c i f i c i t y o f th e enzymes in v o lv e d
in th e s y n th e s is o f DNA. From in fo r m a tio n about th e
e x te n t o f in c o r p o r a tio n o f an an alog a n d /o r i t s a b i l i t y
t o com pete fo r th e s e enzym es i t i s p o s s ib le to make con
c lu s io n s about th e f u n c t io n a l im portance o f ch em ica l
p r o p e r tie s w hich are a lt e r e d by th e abnormal ch em ical
group o f th e a n a lo g . Zamenhof e t al..', ( 8 ) su g g e ste d th a t
th e s iz e and d is t r ib u t io n o f e l e c t r i c a l ch a rg es o f th e
group in th e f i v e p o s it io n o f th e p y rim id in e r in g o f a
thym ine a n a lo g may d eterm in e w hether th e a n a lo g w i l l be
in c o r p o r a te d in t o DNA. A nalogs w ith h a lo g en groups
d if f e r in g in van der W aal's r a d ii and e l e c t r o n e g a t iv it y
have been s tu d ie d . I t would be o f i n t e r e s t t o exam ine
t h i s s u g g e s tio n fu r th e r .
Once i t i s known t h a t an an a lo g i s in c o r p o r a te d
in t o th e DN A o f an organ ism , i t i s p o s s ib le to stu d y i t s
e f f e c t on th e fu n c tio n and p h y s ic a l b eh a v io r o f th e DNA.
I t i s o f i n t e r e s t t o d is c o v e r i f th e p resen c e o f th e
3
analog in DNA w i l l a f f e c t t r a n s c r ip t io n in t o norm al REA
and normal p r o t e in , and i f i t w i l l have a m utagenic e f f e c t ,
presum ably by c a u sin g e r r o r s in r e p l ic a t io n . I t i s a ls o
p o s s ib le t o stu d y v a r ia t io n s i n th e re sp o n se o f c e l l s to
c e r t a in p h y s ic a l a g e n ts , such a s u l t r a v i o l e t and X -ray
ir r a d ia t io n ( 36- 3 9 )* In a d d itio n , th e p rese n ce o f th e
a n a lo g in DBA may a f f e c t th e m eltin g tem perature (3 6 ) or
th e r a te o f in a c t iv a t io n by d ecay ( 4 5 ) .
More g e n e r a lly , p u rin e and p y rim id in e a n a lo g s
have b een o f g r e a t i n t e r e s t b eca u se th e y o f f e r th e
p o s s i b i l i t y o f lim it in g th e r a te o f DNA s y n t h e s is . For
t h i s re a so n th e y have b een e x t e n s iv e ly .stu d ied f o r a n t i
tumor a c t i v i t y .
T h is work was undertaken t o b e g in s t u d ie s o f th e
v a r io u s e f f e c t s o f th e p y rim id in e a n a lo g s m ethylam inp-
d eo x y u rid in e (MAUDR) and d im eth ylam in od eoxyu rid in e
(DMAUDR), s y n th e s iz e d by V is s e r e t a l . , (5 ) and t o
c o n tin u e s t u d ie s o f am inod eoxyu ridin e (AUDR) ( 6 ) . The
ch em ica l g ro u p s, am ino, m ethylam ino and d im eth ylam in o,
p ro v id e a s e r i e s o f s u b s t it u e n t s in th e f i v e p o s it io n o f
th e p y rim id in e r in g . Each i s d if f e r e n t from th e m ethyl
group o f th ym id in e in s i z e and d is t r ib u t io n o f e l e c t r i c a l
c h a r g e s. They th u s seem ed o f s p e c ia l in t e r e s t f o r expand
in g s t u d ie s o f th e s e r i e s o f s u b s t it u e n t s i n t h i s p o s it io n .
In th e c a s e s o f M A U D R and DM AUDR, th e c a lc u la te d r a d iu s o f
th e f i v e s u b s t it u e n t , 3»2A, (9 ) i s s u b s t a n t i a lly g r e a te r
4 -
th a n th e r a d iu s o f th e m ethyl group o f th y m in e, w h ile th e
amino group o f AUD R has s u b s t a n t i a lly th e same r a d iu s a s
th e m ethyl group.
3?o f a c i l i t a t e th e stu d y o f th e e f f e c t s o f th e s e
a n a lo g s we used th e organ ism s E s c h e r ic h ia c o l i (E . c o l i )
15 3f*, d e s c r ib e d by Cohen and Barner (1 1 ) and E. c o l i
K12 0 0 O f, d e sc r ib e d by Ryan ( 1 2 ) , w hich co n ta in e d
prophage A . Both are th y m in e -r e q u ir in g m utants and
E. c o l i 15 3 ? ” h as b een shown to in c o r p o r a te s e v e r a l o th e r
p y rim id in e a n a lo g s in t o i t s DNA (5 0 )*
The p r e s e n t work to be d e s c r ib e d w i l l t e l l o f
s t u d ie s on th e e f f e c t s o f th e s e le c t e d a n a lo g s on th e
grow th o f th e s e o rg a n ism s, and i n th e c a se o f AUDR, o f th e
e f f e c t on th e co m p o sitio n o f th e DNA o f E* c o l i 15 T” .
CH APTER II
HISTORICAL B A C K G R O U N D
S ev er a l s tr u c tu r a l a n a lo g s o f thym ine have been
s y n th e s iz e d , in c lu d in g th e 5-h a lo g en d e r iv a tiv e s o f u r a c il
(1 3 -1 5 ),, 5 -a m in o u ra eil (17) and 5 - th io u r a c il ( 1 8 ). The
n u c le o s id e s and d eo x y r ib o n u c le o sid e s o f many o f th e se
compounds have a ls o been s y n th e s iz e d ( 1 0 , 1 6 , 1 9 -2 0 ).
These a n a lo g s have been found to have v a rio u s
e f f e c t s on th e s y n th e s is and u t i l i z a t i o n o f normal thymine
d e r iv a tiv e s and on th e str u c tu r e and fu n c tio n o f th e D N A
o f v a rio u s organism s. Among th e f i r s t s tu d ie s o f th e se
an a lo g s was th e o b se r v a tio n bt H itch in g s (21) th a t
5 -c h lo r o u r a c il (C1U), 5-brom ou raeil (BU) and io d o u r a c il
(IU) were In h ib ito r y to th e growth o f L a c to b a c illu s
e a s e l i f f o l i c a c id was om itted from th e medium and th e
growth o f th e organism s depended on an exogenous supply
o f thym ine. A d d itio n a l c a se s o f in h ib it io n o f growth o f
L. e a s e l were shown w ith 5-h y d ro x y u ra c il and 5 -a m in o u ra cil
( 2 2 ) . BU was shown to in h ib it th e growth o f S trep to co ccu s
f a e c a l i s by Weygand, Waeker and D ellw eg ( 2 3 ). They a ls o
showed th a t th e BU was in co rp o ra ted in to th e n u c le ic a c id s
o f S . f a e c a l i s , but d id not d if f e r e n t ia t e betw een in c o r
p o r a tio n in to D N A or r ib o n u c le ic a c id .
I t was su b seq u en tly found th a t in most b a c t e r ia l
6
sy stem s th e d e o x y r ib o n u c le o s id e s , b rom odeoxyuridine (BUDR)
and iod id eoxyu rid in e (IUDR) showed g r e a te r a c t i v i t y in
in h i b i t in g c e l l grow th th a n d id th e f r e e b ase or th e
r ib o n u c le o tid e ( 2 4 ) . In v a r io u s mammalian t i s s u e s i t was
shown th a t BUDR and IUDR do n ot i n h i b i t in c o r p o r a tio n o f
la b e le d form ate in t o a c id - s o lu b le thym ine n u c le o t id e s but
do in h i b i t su b seq u en t in c o r p o r a tio n o f thym ine compounds
in t o DNA ( 2 5 - 2 7 ) . T h is su g g e ste d th a t th e s e a n a lo g s may
b lo c k p h o sp h o r y la tio n or p o ly m e r iz a tio n o f thym ine
n u c le o t id e s .
In 1 9 5 4 , Gohen and Barner (7 ) d e sc r ib e d a th ym in e-
l e s s (T~) s t r a in o f E. c o l i , d e sig n a te d 15 T” , w hich in
th e ab sen ce o f thym ine e lo n g a te s but f a i l s to d iv id e and
" d ies" r a p id ly . T h is l o s s o f a b i l i t y t o form c o lo n ie s
was a t tr ib u t e d to unbalanced grow th, in v o lv in g norm al RNA
and p r o t e in s y n th e s is but d e f i c i e n t DNA s y n t h e s is . They
r e p o r te d t h a t BU can p a r t i a l l y r e p la c e th e thym ine r e q u ir e
ment i n such a m utant, p e r m ittin g a tem porary in c r e a s e in
v ia b le c e l l number. In th e same y e a r , b o th Dunn and Sm ith
(2 8 ) and Zamenhof and G r ib o ff (2 9 ) r e p o r te d th e in c o r p o r a
t i o n o f BU in t o th e DNA o f E. c o l i s t r a i n s . The l a t t e r
ex p erim en ters u sed T~ m utants w h ile Dunn and Sm ith u sed a
su lp h a n ila m id e medium to in d u ce tem porary thym ine d e f ic ie n
c y , th u s en h ancing th e p o s s i b i l i t y o f a n a lo g in c o r p o r a tio n .
Dunn and Sm ith a ls o r e p o r te d th e in c o r p o r a tio n o f BU and
IU in to DNA o f th e b a c te r io p h a g e s T2 and T^ ( 3 0 ) . The
?
In c o r p o r a tio n o f 5 - c h lo r o u r a c il (G1U) and IU in t o th e DNA
©f thym ine r e q u ir in g m utants o f E. c o l i was r e p o r te d by
Zamenhof e t a l . , ( 9 ) . These, w orkers a ls o r e p o r te d th a t
BU exchanged w ith thym ine i n r e s t in g c e l l su sp e n sio n s t©
g iv e 8 -4 8 p er ce n t rep la cem en t o f t o t a l; thym ine i n th e
1NA. Dunn and Sm ith (3 0 ) showed th a t th e in c r e a s e ©f BU
c o n te n t o f th e DNA was accom panied by d ecr ea se d thym ine
c o n te n t, p ro v in g d i r e c t l y th a t BU r e p la c e d th ym in e. They
a ls o d em on strated t h a t i n h i b i t i o n o f grow th co u ld be
r e v e r s e d by th e a d d itio n ©f th y m id in e , e x c e p t fo r i n h i b i
t i o n by C1UDR, f o r w hich u r a c il a ls o had t© be added in
©rder t o r e v e r s e grow th in h i b i t i o n . T h is f a c t r e f l e c t e d
more th an one s i t e o f a c t i o n .f o r G1UDR. Dunn h as shown
th a t G1U i s in c o r p o r a te d t o a sm a ll e x te n t in t o RNA in
I - c o l l 15 T~ ( 3 1 ) .
In c o n s id e r in g th e e x te n t o f in c o r p o r a tio n o f th e
v a r io u s a n a lo g s s tu d ie d i n E. c o l l s tr a in . I , Zamenhof
e t a l . , ( 8 ) c o n sid e r e d t h a t th e e x te n t o f in c o r p o r a tio n
o f an a n alog in t o DNA m ight be r e la t e d to th e s iz e and
d is t r ib u t io n o f e l e c t r i c a l ch a rg es o f th e group i n th e
f i v e p o s it io n o f th e p y rim id in e r in g . They p o in te d out
t h a t i n s t r a in I , BU i s in c o r p o r a te d to a g r e a te r e x te n t
th a n G1U, aad G1U i s in c o r p o r a te d to a g r e a te r e x te n t th an
IU . The brom ine atom i n BU h as th e van d er W aal's r a d iu s ,
O - •
1 .9 5 A , w hich i s most s im ila r t© 2.0A th e r a d iu s o f th e
m ethyl group o f th y m in e. The r a d iu s f o r G1U i s 1.8A and
8
O
f o r IU th e r a d iu s i s 2 .1 5 A . F iv e f lu o r o u r a c il (FU) i s n ot
found t o be in c o r p o r a te d in to DNA. The f lu o r o group h as a
O
r a d iu s o f 1 .3 5 A , w hich i s v e r y s im ila r t o th e r a d iu s o f
o
th e hydrogen in u r a c i l , 1 .2 A , and FU i s found t o be in c o r
p o ra ted in t o RNA ( 3 2 ) . However, Dunn and Sm ith r e p o r te d
th a t th e amount o f in c o r p o r a tio n o f th e h a lo g en a te d
u r a c i ls i s n o t c o r r e la te d w ith s iz e o f th e h a lo g en i n th e
c a se o f E. c o l i 13 T~ (3 0 )* They found th a t i n t h i s
organism C1U was in c o r p o r a te d to a much g r e a te r e x te n t
th a n BU or IU. They su g g e s t th a t th e amount o f in co r p o r a
t io n o f th e h a lo g en a te d u r a c i l s depends v e r y much on
grow th c o n d itio n s and th e p a r t ic u la r b a c t e r i a l s t r a in
b e in g s tu d ie d .
A nalogs w hich have b een found to be in c o r p o r a te d
in t o DN A have g e n e r a lly b een found to be m u tagen ic. L i t -
man and P ardee (3 5 ) o b serv ed th e appearance o f a h ig h
p r o p o r tio n o f m utants i n T even phages grown i n th e
p resen ce o f BU, C1U, IU and BUDR. A d d itio n o f th ym id in e
t o th e grow th medium p rev e n ted th e appearance o f th e s e
m u tan ts. From a s e r i e s o f k in e t ic s t u d ie s o f th e e f f e c t s
o f BUDR on T2 developm ent th e y were a b le t o show th a t BUDR
was n ot e f f e c t i v e a s a mutagen u n t il th e tim e when phage
DNA s y n t h e s is b egan . They a ls o showed t h a t when BUDR i n
th e medium i s r e p la ced by th ym id in e soon a f t e r m u ta g en esis
b e g in s , m utants co n tin u e to appear a s an in c r e a s in g p er
ce n ta g e o f th e p h a g es. They su g g e ste d t h a t m u ta tio n s are
9
induced. b o th in th e p r o c e s s o f in tr o d u c tio n o f BU in t o
phage DNA and d u rin g th e r e p l ic a t io n o f DNA c o n ta in in g BU.
In o th e r words BU m ight be in c o r p o r a te d by p a ir in g w ith
e it h e r gu anin e or a d e n in e , and th e n m ight f a i l to p a ir in
th e same manner in th e n e x t d u p lic a tio n . Thus assum ing
th a t BU sh o u ld n o rm a lly p a ir w ith a d e n in e , th e r e are two
p o s s ib le ways in w hich th e r e would be an e v e n tu a l s u b s t i
t u t io n o f one b a se p a ir f o r a n o th er; a m istak e in p a ir in g
d u rin g in c o r p o r a tio n , or a m istak e d u rin g d u p lic a tio n .
In b o th c a s e s th e u ltim a te r e s u l t w ould be an a lt e r e d b ase
seq u en ce. Such a scheme would exp la i n BU in d u ced muta
t i o n s a s ch em ica l changes i n th e DNA.
A nother e f f e c t o f in c o r p o r a te d a n a lo g s on b a c t e r ia
( 2 9 , 3 0 ) phage ( 3 3 ) and mammalian c e l l s (th e human c e l l
l i n e D98S c u lt iv a t e d i n v i t r o ( 3 6 ) ) i s th e p ro d u c tio n o f
an in c r e a s e d s e n s i t i v i t y to u l t r a v i o l e t ir r a d ia t io n . The
e x te n t o f in c r e a s e d s e n s i t i v i t y i s shown to be p a r a lle le d
by e x te n t o f in c o r p o r a tio n , BU h avin g a g r e a te r e f f e c t
th a n IU and C1U. I t h as a ls o b een shown (34-) th a t t h i o -
g u a n in e, w hich i s in c o r p o r a te d to a sm a ll e x te n t in
E. c o l i en h ances th e s e n s i t i v i t y o f E. c o l i to u ltr a v io le t
i r r a d ia t i o n , w h ile a n a lo g s such a s FU and 2 -th io th y m in e
w hich a re n o t in c o r p o r a te d in t o DNA do n o t a f f e c t th e
u l t r a v i o l e t s e n s i t i v i t y o f b a c t e r i a l c e l l s . Hanawalt
and S etlo w (3 9 ) ob served a d e la y i n in c o r p o r a tio n o f
32
n u c le o tid e in t o DNA fo llo w in g ir r a d ia t io n w ith UV.
10
T h is agreed w ith p r e v io u s ev id en ce th a t DNA s y n th e s is i s
th e c e l l u l a r fu n c tio n c h i e f l y e f f e c t e d by UV ir r a d ia t i o n .
They found th a t dark r e c o v e r y mechanisms e v e n tu a lly
r e s t o r e s y n t h e s is to i t s normal r a t e . However th e D N A
produced may n o t be norm al.
V ariou s p h o to p ro d u cts o f UV ir r a d ia t io n o f th e
fo u r normal DNA com ponents have b een d e s c r ib e d ( 3 8 - 4 2 ) .
Among such known p h o to p ro d u cts are thym ine dim ers and th e
h y d r a tio n p ro d u cts o f c y t o s in e and u r a c i l , produced e it h e r
by th e h y d r o ly tic s p l i t t i n g o f th e p y rim id in e r in g or by
th e a d d itio n o f w ater a c r o s s th e 3 -6 double bond. Evidence
has been g iv e n f o r an enzym ic p h o to r e a c tiv a tio n sy stem (4])
w hich presum ably rem oves or b reak s down p h o to p r o d u c ts,
p r in c ip a ll y thym ine d im ers. I t has b een su g g e ste d th a t
when DNA s y n t h e s is i s r e s to r e d th e p r o b a b ilit y o f a copy
e r r o r p er DNA stra n d w i l l be p r o p o r tio n a l t o th e amount o f
photop.roduct p r e s e n t a t th e tim e o f i t s r e p l i c a t i o n ( 4 2 ) .
L o rk iew icz and S z y b a lsk i (4 3 ) r e p o r te d th a t th e r e
i s im pairm ent o f th e p h o to r e a c tiv a tio n o f BU c o n ta in in g
b a c t e r ia . They su g g e st t h a t th e p r esen c e o f BU i n th e
DNA may e it h e r ca u se ir r e v e r s i b l e r a d ia t io n damage or i t
may p rev en t th e r e p a ir o f n o rm a lly r e v e r s ib le r a d ia t io n
damage. Wacker e t a l . , (4 4 ) r e p o r te d t h a t th e p r in c ip a l
e f f e c t o f u l t r a v i o l e t ir r a d ia t io n on BU i s d eb rom in ation
to u r a c il or h y d r o x y u r a c il.
__________The p re se n ce o f BUDR, C1UDR and IUDR in th e DNA
Df E. c o l i ( 5 5 ) and c u ltu r e d mammalian c e l l s (4 5 ) has been
shown to ca u se in c r e a s e d s e n s i t i v i t y to X -ra y s. E rick so n
and S z y b a lsk i (4 5 ) found t h a t IUDR produced a g r e a te r
s e n s i t i v i t y to X -ir r a d ia tio n th a n BUDR or C1UDR.
Ragni and S z y b a ls k i (4 5 ) found th a t th e p re sen ce
o f 01UDR, IUDR, and BUDR i n th e DEA o f mammalian c e l l s
-52
in c r e a s e s th e P^ d ecay s e n s i t i v i t y . They showed th a t
x?
th e enhancem ent o f s e n s i t i v i t y to l e t h a l e f f e c t s o f
d eca y r e q u ir e s th a t th e h a lo g en atom s be in c o r p o r a te d in t o
52
th e stran d o p p o s ite to th a t b ea rin g th e P atom s. They
ta k e t h e ir d a ta t o in d ic a t e th a t th e l e t h a l ev e n t i s a
s c i s s i o n th rou gh b o th str a n d s o f th e DEA m o lecu le and th a t
th e h a lo g en l a b i l i z e s th e phosp h ate e s t e r bond by i t s e le c
t r o s t a t i c in t e r a c t io n w ith th e phosp h ate atom , th u s
enhancing th e p r o b a b ilit y o f th e s c i s s i o n e v e n t.
The p r esen ce o f an an a lo g in DEA m ight c o n c e iv a b ly
a f f e c t th e m e ltin g tem p era tu re. S z y b a lsk i (5 6 ) r e p o r te d
th a t th e m e ltin g tem p eratu re o f B ,U D R c o n ta in in g D M A i s
h a rd ly d if f e r e n t :from normal DEA; 8 5 .8 °C fo r normal DEA
and 8 5 .4 ° C f o r BUDR DEA.
A nother p o s s ib le e f f e c t o f an a n a lo g on DEA s tr u c
tu r e and fu n c tio n i s i l l u s t r a t e d by th e r e p o r t o f Dunn
and. Sm ith th a t w h ile most b a c t e r ia c o n ta in a sm all p ro
p o r tio n o f 6-m eth ylam in op u rin e as a norm al c o n s t it u e n t o f
t h e i r DEA ( 4 7 ) , E. c o l i 15 T~ grown i n th e ab sen ce o f
thym ine or i n th e p resen ce o f 5-a m in o u r a c il or
12
2 -th io th y m in e lias a much in c r e a s e d 6 -m eth ylam inop u rin e
co n te n t ( 4 8 ) . They found no ev id en ce th a t th e 5 -am in o-
u r a c il was in c o r p o r a te d in t o th e DNA. T h is DNA, h ow ever,
had an in c r e a s e in 6 -m eth ylam inop u rin e co n ten t e q u iv a le n t
to about 15 p er c e n t o f th e ad en in e and th u s a t o t a l
in c r e a s e in p u rin e c o n te n t, and a d ecr ea sed thym ine
c o n te n t. Such DNA can n ot be r e c o n c ile d w ith th e W atson-
C rick model o f th e DNA s tr u c tu r e and i s c o n sid e r e d t o be
abnorm al, n o n -fu n c tio h a l DNA. T h ie l and Zamenhof (4 9 )
14
have shown th a t m ethionine-C H ^-C can se rv e a s a m ethyl
donor i n th e fo rm a tio n o f 6 -m eth ylam inop u rin e and th a t
th e p r esen ce o f a m in o u ra cil p a r t i a l l y in h ib it e d th e
uptake o f exogenous m eth io n in e in E. c o l i 15 T ” . They
a ls o r e p o r te d th a t th e r e was a co n tin u ed in c r e a s e i n th e
amount o f 6 -m eth ylam in op u rin e i n th e DN A even a f t e r n e t
DNA s y n t h e s is had c e a s e d . They found th a t such DN A
r a p id ly l o s t 6-m eth ylam inop u rin e a f t e r tr a n s fe r to a
normal medium w ith o u t a m in o u r a c il. In one hour th e
6 -m eth ylam inop u rin e c o n te n t d ecr ea se d by 28 p er ce n t
w h ile DNA c o n te n t had in c r e a s e d by 10 p er c e n t .
c o l i 15 T“ th e in tr o d u c tio n o f IU , BU, or
C1U in t o th e DNA was accom panied by an in c r e a s e in
6 -m eth ylam inop u rin e c o n te n t ( 5 0 ) . The mechanism by w hich
grow th in th e absence o f th y m in e, grow th in th e p r esen c e
o f a m in o u r a c il, h a lo g en a te d u r a c i l s , or 2-th io th y m in e a l l
i nduce th e m e th y la tio n o f ad en in e to 6-m eth ylam inop u rin e
13
i s unknown.
S tu d ie s o f A m in o u ra c il. A m inou racil was f i r s t
s y n th e s iz e d in 1909 "by W heeler and Johnson ( 1 7 ) .
H itching® e t a l . , showed th a t a m in o u ra cil was in h ib it o r y
t o th e grow th o f L. c a s e i ( 2 2 ) . S m ith, R u s s e ll and
Kugelman ( 5 0 ) re p o r te d th a t a m in o u ra cil su p p ressed a l l
d iv is io n o f r o o t t i p c e l l s o f V ic ia fa b a . Removal o f
th e s e c e l l s from an a m in o u ra c il medium r e s u lt e d in
*
p a r t i a l l y sy n ch ro n ized d iv is io n o f e e l l s .
R ob erts and V is s e r (5 1 ) showed th a t 5 -a m in o u ri-
d in e (AUR) in h ib it e d th e grow th o f w ild ty p e H. c r a s s a
and a p y rim id in e r e q u ir in g s t r a in 1298. AUR has a ls o
b een shown t o in h i b i t th e p ro p a g a tio n o f T h e ile r 's GD.VII
v ir u s ( 5 2 ) and th e grow th o f v a r io u s mouse tum ors ( 5 3 ) .
AUR has b een shown t o i n h i b i t in c o r p o r a tio n o f la b e le d
form ate and carbam yl a s p a r ta te in t o RNA p u r in e s and
p y r im id in e s o f l i v e r n u c le ic a c id s in r a t s b ea r in g human
tumor tr a n s p la n ts HS#1 C5^)«
In 1 9 5 7 , B e ltz and V is s e r s y n th e s iz e d am inodeoxy
u r id in e (AUDR) a s a p o s s ib le in h ib it o r o f p y rim id in e
n u c le o tid e b io s y n t h e s is or fu n c tio n ( 1 9 ) . They showed
th a t AUDR had a stro n g in h ib it o r y e f f e c t on th e grow th
IL. c o l i K 1 2 . T h is in h i b i t i o n c o u ld be c o m p le te ly
r e v e r se d by th ym id in e and p a r t i a l l y r e v e r s e d by u r id in e ,
d e o x y u r id in e , c y t id in e and d e o x y c y tid in e . Thymidine th u s
appeared to be th e p rod u ct o f th e m e ta b o lic b lo c k , w h ile
14
th e n u c le o s id e s appeared to he p r e c u r s o r s . I t was p ro
p osed th a t AUDR i n h i b i t s th e m e th y la tio n o f d eo x y u rid in e
monophosphate (dUMP) to th ym id in e monopho'sphate (T IP ).
P r ie d la n d and V is s e r (5 5 ) showed th a t grow th in th e
p resen ce o f AUDR and an exogenous sou rce o f thym ine to
r e s t o r e norm al grow th, in h ib it e d th e in c o r p o r a tio n o f
14
is o t o p e from 3 - C s e r in e in t o DN A thym ine in E. c o l i K12.
The in c o r p o r a tio n o f is o t o p e in t o DNA ad en ine was n ot
e f f e c t e d how ever. T h is was in te r p r e te d a s fu r th e r evidence
th a t AUD R i n h i b i t s th y m id y la te s y n th e ta s e , th e enzyme which
b r in g s about th e m e th y la tio n o f dUM P to TMP. In a d d it io n ,
AUDR p o te n t ia t e d th e in h ib it o r y e f f e c t o f FUDR a t concen
t r a t io n s o f th e l a t t e r w hich i n h i b i t th y m id y la te synth etase
' ' T j , .
but n ot th e m etabolism o f RNA p r e c u r s o r s . T his in d ic a te d
th a t AUDR i n h i b i t s th e m etabolism o f thym ine compounds
su bseq uent t o th e fo rm a tio n o f TMP. AUDR th u s was shown
t o i n h i b i t th e s e q u e n tia l r e a c t io n s in v o lv in g th y m id y la te
fo rm a tio n and u t i l i z a t i o n o f th y m id y la te .
By a n a lo g y w ith o th e r n u c le o s id e d e r iv a t iv e s
w hich a c t as in h ib it o r s and have b een found to b e co n v er
te d to th e n u c le o tid e ( 5 6 ; 5 7 ) , P r ie d la n d and Y is s e r su g
g e s te d th a t AUDR a ls o may be co n v erted t o th e n u c le o tid e
b e fo r e i t fu n c tio n s as an i n h i b i t o r . However, a t th e
tim e th e r e was no ev id e n c e f o r n u c le o tid e fo rm a tio n .
CHAPTER I I I
EXPERIMENTAL
B a c te r ia l S tr a in s - E s c h e r ic h ia c o l i K 12 (T was
o b ta in ed from Dr. M argeret L ie b , U n iv e r sity , o f Southern
C a lif o r n ia . E. c o l i 15 T~ was o b ta in ed through th e
c o u r te s y o f Dr. R. Romig, U n iv e r s ity o f C a lifo r n ia a t
Los A n g e les.
S y n th e tic Growth Medium - A m o d ifie d Gray and Tatum 's
medium ( 5 8 ) was p rep ared by d is s o lv in g 1 0 .0 g HH^Cl,
2 .0 g NH^NOj, 4 .0 g Na2S04 , 6 .0 g K 2HP04 , 2 .0 g KB^PO^,
2 .0 g MgSO^ • 7H20 , and 0 .2 cc o f a 10 p er c e n t s o lu t io n
o f CaCl2 p er one l i t e r o f w a te r. T h is s o lu t io n was
supplem ented w ith 2 0 .0 ml o f a s t e r i l e 10 p er c e n t s o lu
t io n o f c a s e in h y d r o ly s a te , and 1 .0 ml o f a s o lu t io n o f
tr a c e ele m en ts p er m l. The s o lu t io n was th e n a u to c la v e d
a t 15 lb s p r e ssu r e fo r tw en ty m in u tes. A fte r c o o lin g ,
2 0 .0 ml o f a s t e r i l e 20 p er c e n t s o lu t io n o f g lu c o s e was
added.
S to ck C u ltu re s - The E. c o l i s t r a in s were sto r e d on an
agar s la n t m ain tain ed a t 0°C. S tock c u lt u r e s were grown
i n try p to n e b ro th w ith v ig o r o u s a e r a tio n and s to r e d a t
0°C f o r up to th r e e w eeks. S tock c u lt u r e s were used to
prep are in o c u la f o r in d iv id u a l ex p er im en ts.
In o c u la - In o c u la were p rep ared by adding one ml o f sto c k
15
16
c u ltu r e p er 50 ml o f s y n th e t ic grow th medium supplem ented
w ith 5 U jg th ym id in e p er m l. The c u lt u r e s were in cu b a ted
a t 37°C i n a r e c ip r o c a tin g shaker fo r from fo u r to f i v e
hours u n t i l t u r b id it y rea ch ed about 0 . 1 5 , a s read on th e
Coleman c o lo r im e te r a t 390 m u *. An a liq u o t o f th e c u ltu r e
was d ilu t e d f o r a s s a y on tr y p to n e a g a r. The c o n c e n tr a tio n
8
o f v ia b le b a c t e r ia was u s u a lly found to be betw een 2x 10
Q
and 3x10 p er m l. T h is c u ltu r e was c e n t r ifu g e d , th e
■ * 1
p e l l e t washed in c o ld s a lin e s o lu t io n and r e c e n tr ifu g e d .
The f i n a l p e l l e t was resu sp en d ed i n c o ld s y n th e t ic grow th
v ' ■ ' ■
medium to g iv e an a p p r o p r ia te c o n c e n tr a tio n o f b a c te r ia
p er m l. T h is su sp e n sio n o f c e l l s was th e n used as an
inoculum f o r a s in g le grow th ex p erim en t.
Growth E xperim ents - A s e r i e s o f 300 ml E rlenm eyer f l a s k s
was p rep a red , each c o n ta in in g 50 ml o f s y n th e t ic grow th
medium. One such f l a s k , th e maximal th ym id in e c o n t r o l,
was supplem ented w ith 0 .5 ml o f a 0 .1 p er c e n t s o lu t io n o f
th ym id in e (o r thym ine) g iv in g 10 q g /m l. A secon d f l a s k
th e m inim al th ym id in e c o n t r o l, was supplem ented w ith 0 .5
ml o f a 0 .0 1 p er c e n t s o lu t io n o f th ym id in e g iv in g 1 m jg/dal.
The rem ainder o f th e f l a s k s were a ls o supplem ented w ith
t h i s m inim al amount, o f th y m id in e. In a d d itio n each was
supplem ented w ith th e p a r t ic u la r a n a lo g to be s tu d ie d .
In g e n e r a l, c o n c e n tr a tio n s o f a n a lo g v a r ie d from 10 ug/m l
t o 100 q g /m l.
T h is s e r i e s o f f l a s k s was in o cu la ted , w ith 1 ml o f
17
7
th e in ocu lu m to g iv e a p p ro x im a tely 2x 1 0 1 b a c t e r ia p er m l.
The c u lt u r e s were th e n in c u b a ted a t 37°C in th e r e c ip r o c a
t in g sh a k er. A liq u o ts o f a l l c u lt u r e s were ta k en a t one
hour and th e n one h a lf hour i n t e r v a l s . They were rea d a t
390 m i} on th e Coleman c o lo r im e te r , and a ssa y e d on try p to n e
agar fo r th e c o n c e n tr a tio n o f v ia b le c e l l s .
S y n th e s is o f R a d io a c tiv e AUDR - 250 q c u r ie s - o f 2 - ^ C
d eo x y u rid in e p urch ased from C a lifo r n ia C orp oration fo r
B io ch em ica l R esea rch , was d is s o lv e d in. 3 a l o f w ater in a
t h ic k w a lle d t e s t tu b e . To t h i s s o lu t io n 448 mg (0 .0 0 1 9 9
m oles t o t a l ) o f d e o x y u rid in e were added. The s o lu t io n was
^'
th e n fr o z e n and ly o p h i liz e d . To th e d ry ly o p h iliz e d s o lid ,
a c e t i c anhydride and eq u im olar brom ine w ere added, a cco rd
in g t o th e method o f P r ie d la n d and V is s e r f o r th e
s y n th e s is o f AUD R (5 5 )* The r e a c t io n was a llo w e d t o p ro
ceed f o r one and one h a lf h o u r s. The r e s u lt in g s o lu t io n
o f a c e ty la te d 5-brom od eoxyu rid in e was t r e a t e d i n a manner
s l i g h t l y d if f e r e n t from t h e ir m ethod. I t was ev a p o ra ted
under g e n t le s u c tio n from an ;a s p ir a to r . E th anol was th en
added and ev a p o ra ted under a stream o f a i r . T h is s te p
was r e p e a te d u n t i l w h ite c r y s t a l s were form ed. A v is c o u s
la y e r o f dark orange e th a n o l was rem oved. The c r y s t a l s
were l e f t o v e r n ig h t in a vacuum d e s s ic a t o r over NaOH
p e l l e t s .
The method o f F r ie d la n d and V is s e r was th e n
fo llo w e d f o r th e s y n th e s is o f AUDR from a c e ty la te d
18
b rom od eoxyu rid in e. The r e s u lt a n t AUDR was p u r if ie d by
d escen d in g chrom atography on Whatman 3m m p a p er. B u ta n o l,
e th a n o l, w a te r , 5 0 :3 0 :2 0 , was th e chrom atographic s o lv e n t .
The s t r i p s o f AUDR were i d e n t i f i e d by th e appearance o f a
y e llo w c o lo r ( c h a r a c t e r i s t i c o f a m in o u ra cil and i t s d e r i
v a t i v e s ) when th e paper was sprayed w ith a s o lu t io n o f
d im eth ylam in ob en zald eh yd e, a s d escrib ed , by F rie d la n d (5 9 )•
The s t r i p s were c u t o u t, and r e p e a te d ly e lu te d w ith 0 .1
R HC1. The a c id s o lu t io n was c o n c e n tr a te d on th e r o ta r y
' ; i - ■
ev a p o ra to r u n t i l pure w h ite c r y s t a l s ap peared . These were
c o l l e c t e d and w ashed. T h eir chrom atographic p u r ity was
proven by chrom atography on S c h le ic h e r and S c h u e ll orange
B p a p er, # 5 8 9 , u sin g b u ta n o l, e th a n o l * w a te r , 5 0 :3 0 :2 0 , a s
th e s o lv e n t . A s in g le u l t r a v i o l e t a b so rb in g sp o t moved to
th e same p o s it io n a s a c o n tr o l sp o t o f a u th e n tic AUDB..
T h is sp o t was shown t o corresp on d to th e p o s it io n o f th e
o n ly r a d io a c tiv e peak .
14
grow th o f B a c te r ia in Medium C on tain in g 2 - C AUDB - One
hundred and e ig h ty mg o f AUDR, s p e c i f i c a c t i v i t y 1.02x10^
icpm/n m ole, was d is s o lv e d in 21 ml s t e r i l e H? 0 . T h ir ty
! ' |
jm g o f AUDR were added and th e pH was th e n a d ju ste d to about
|7 .0 . An inoculum o f B. c o l i 15 T~ was p rep a red . The cells!
! 9 '
iwere resu sp en d ed to a f i n a l c o n c e n tr a tio n o f 5x 10 bacteriAj
i i
!per m l. Four ml o f t h i s su sp e n sio n were in o c u la te d in t o |
I
leach o f se v en fo u r l i t e r Brlenm eyer f l a s k s , each c o n ta in
in g one l i t e r o f s y n th e t ic growth medium, 1 ml o f a 0 .1
19
p er ce n t s o lu t io n o f th ym id in e (1 t^g/ml) and th r e e ml o f
1A
th e 1 p er c e n t s o lu t io n o f 2 - C AUDE (3 0 q g /m l).
The f l a s k s were in cu b a ted a t 37°C on th e r e c ip r o
c a tin g sh a k er. A liq u o ts were removed, f o r t u r b id it y
r e a d in g s a t one hour and th e n f i f t e e n m inute in t e r v a ls
d u rin g th e n e x t hour. A fte r two hours th e c u lt u r e s had
b een shown p r e v io u s ly t o corresp on d t o a c o n c e n tr a tio n o f
Q
b a c t e r ia o f about 2x10 . The f l a s k s were r a p id ly c h i l l e d
and s to r e d o v e r n ig h t a t 0 ° C .- An a liq u o t was d ilu t e d f o r
a ssa y on tr y p to n e a g a r.
The c u ltu r e was c e n tr ifu g e d a t 7000 rpm f o r tw en ty
m in u tes i n th e S e r v a ll c e n t r if u g e . The c le a r su p ern a ta n t
was d eca n ted and th e c e l l s were resu sp en d ed in 2 /5 volume
o f 0 .1 4 M MaCl and r e c e n t r if u g e d . The p e l l e t s were washed
a g a in 2 /5 volum e o f saline-E D T A s o lu t io n ( 0 .1 5 M FaCl and
.1 M EDTA) a cc o rd in g t o Marmur ( 6 0 ) . The p e l l e t s were
com bined and th e wet packed c e l l s , w eig h in g a p p ro x im a tely
6 g , were suspended i n 67*5 ml saline-E D T A a cc o rd in g to
Marmur ( 6 0 ) .
P r e p a r a tio n o f DNA - F iv e ml o f a 25 p er c e n t s o lu t io n o f
sodium la u r y l s u lf a t e was added to th e saline-E D T A su sp en
s io n o f b a c t e r ia . The m ixtu re was h e a te d a t 6 0 ° f o r te n
m in u te s, and c o o le d to room tem p era tu re. Two volum es o f
100 p er c e n t e th a n o l were added. The r e s u lt in g p r e c ip i
t a t e was washed in 60 p er c e n t EtOH and suspended i n 60 ml
1 .5 M NaCl, 0 .1 5 M tr iso d iu m c i t r a t e . __________________________
20
T h is s o lu t io n was c e n tr ifu g e d a t 19*000 rpm fo r t h i r t y
m in u tes i n a Spinco U lt r a c e n t r ifu g e . The su p ern a ta n t was
d eca n te d . The p r e c ip it a t e was resu sp en d ed i n 40 ml o f
0 .1 5 M NaCl, 0 .0 1 5 M tr iso d iu m c i t r a t e and th e c e n t r if u
g a t io n p r o c e ss r e p e a te d . Both su p e rn a ta n ts were p o o led
and two volum es o f e th a n o l were added. The r e s u lt in g
p r e c ip it a t e was d is s o lv e d i n 40 ml o f 0 .1 5 M NaCl, 0 .0 1 5 M
sodium c i t r a t e and c e n tr ifu g e d a t 19,000 rpm fo r 30
m in u tes. The su p ern a ta n t was d eca n ted and p r e c ip it a t e d
w ith two volum es o f e th a n o l. The p r e c ip it a t e d n u c le ic
a c id s were d is s o lv e d in 15 ml o f 0 .1 5 M sodium c i t r a t e
b u f f e r , pH 7 .3 *
P u r if ic a t io n o f D M A - Removal o f P r o te in - A sam ple o f
50 mg o f t r y p s in o b ta in ed from C a lifo r n ia C orp oration fo r
B io ch em ica l R esearch was tw ic e p u r if ie d a cc o rd in g t o th e
method o f Dunn and Sm ith (4 8 ) and th e f i n a l p r e c ip it a t e
d is s o lv e d i n 0 .5 ml ^ 0 . Of t h i s s o lu t io n 0 .2 ml was
added to th e s o lu t io n o f n u c le ic a c id s in sodium c i t r a t e
b u f f e r . The s o lu t io n was in c u b a ted a t 37° C fo r e ig h te e n
h o u r s. A fte r t h i s tim e an eq u a l volum e o f 0 .1 M MgSO^
and a c e t i c a c id t o b rin g th e pH b elow 6 .0 were added.
i
Twice th e r e s u lt in g volum e o f e th a n o l was th en added, and j
th e p r e c ip it a t e d n u c le ic a c id s were d is s o lv e d i n 18 ml o f j
0 .0 1 5 M NaCl, 0 .0 0 1 5 M tr iso d iu m c i t r a t e . Two ml o f 1 .5 M
NaCl, 0 .1 5 M sodium c i t r a t e were added.
Removal o f RNA - Marmur's method (6 0 ) was u sed . A 20 p er
21
c e n t s o lu t io n o f r ib o n u c le a s e (REAase) in .1 5 M EaC.1, pH
5 , was h ea ted a t 8 0 ° f o r te n m in u tes t o remove d eo x y rib o
n u c le a se (DNAase) a c t i v i t y . To th e s o lu t io n o f n u c le ic
a c id s , >50 ml o f REAase s o lu t io n were added. The s o lu t io n
was in cu b a ted a t 37° fo r 4-5 m in u tes. At th e end o f t h i s
tim e two volu m es o f e th a n o l were added. The n u c le ic a c id s
were c o l l e c t e d by e e n t r if u g a t io n a t 4000 rpm and d is s o lv e d
i n 18 ml o f 0 .0 1 5 M HaCI, 0 .0 0 1 5 M sodium c i t r a t e . To
t h i s s o lu t io n 2 .1 1 ml o f acetate-ED T A ( .1 5 M NaCl and 0 .1
M e th y le n e d ia m in e te tr a a c e ta te , pH 8 ) s o lu t io n were added,
and w h ile r a p id ly s t i r r i n g w ith a m agnetic s t i r r e r , 1 1 .3 9
ml ( .5 4 volu m es) o f is o p r o p y l a lc o h o l were added d ro p w ise.
The r e s u lt in g s o lu t io n was c e n t r ifu g e d , th e p r e c ip it a t e d
DEA removed and a few a d d it io n a l ml o f is o p r o p y l a lc o h o l
added to th e su p e r n a ta n t, w hich was th en r e c e n tr ifu g e d
tw ic e to y i e l d more p r e c ip it a t e d DEA. The com bined p r e c i
p i t a t e s were d is s o lv e d i n 7*0 ml o f .0 0 2 M MgSO^.
H y d r o ly sis o f DEA - The method o f Dunn and Sm ith was used
( 4 8 ) . A s o lu t io n o f 4 mg o f d eo x y r ib o n u c le a se in 4 ml
o f H 2 O was prepared.. Of t h i s s o lu t io n 0 .2 5 ml were added
t o th e s o lu t io n o f DEA i n 0 .0 0 2 M MgSO^. The m ixtu re was
th e n m a in ta in ed a t 37°C and pH 7*6 f o r s i x h o u r s, by means
o f an a u to m a tic t i t r a t o r (R a d io m eter). The t i t r a n t was
0 .0 5 m EH^OH. A fte r s i x hours an eq u a l volume o f 0 .0 4 M
g ly c in e b u f f e r , pH 9 - 2 , and 0 .1 5 ml o f p u r ifie d snake
venom p h o sp h o d ie ste r a se (o b ta in e d from C a lifo r n ia
22
C orp oration fo r B io ch em ica l R esearch ) were added. The
s o lu t io n was in cu b a ted a t 37°C f o r f i v e h o u r s, w h ile th e
pH was m ain tain ed a t betw een 9*0 and 9 .6 by o c c a s io n a l
a d d itio n s o f .0 5 M N H 4 0H.
S e p a r a tio n o f N u c le o tid e s from H ydrol.ysate o f DNA-
644 m oles o f a m in o u rid in e-*5'-m onophosphate (p rep a red by
p h o sp h o r y la tio n o f am in ou rid in e w ith H^PO^ and '£ ’ 2 ®^
a cco rd in g to a m o d ific a tio n o f th e method o f Ik eh a ra ,
Ueda, and Ikeda ( 6 1 ) ) were added and th e h y d r o ly sa te was
p la c e d on a column o f D o w ex -l-fo rm a te, 100-2 0 0 m esh, w hich
was a p p ro x im a tely 20 cm lo n g and 1 .3 cm in d ia m e te r. The
column was washed w ith w ater u n t i l th e e lu a te c o n ta in e d
no more u l t r a v i o l e t a b so rb in g m a te r ia l. The column was
d ev elo p e d by an exten d ed g r a d ie n t e lu t io n p rocedu re as
d e sc r ib e d by Murakami e t a l . , ( 6 2 ) , u sin g a r e s e r v o ir o f
1 l i t e r and a s o lu t io n o f 1 .5 N form ic a c id . T h is s o lu
t io n was changed t o 9 N form ic a c id a f t e r th e e l u t io n o f
d eoxyad en osin e m onophosphate (dAMP). F r a c tio n s o f about
fo u r ml were c o lle c te d ..
A n a ly s is o f E th anol S o lu b le F r a c tio n o f B a c te r ia l ILysate
f o r A cid S o lu b le s - The o r ig in a l e th a n o l p r e c ip it a t io n o f
n u c le ic a c id s from ly s e d c e l l s y ie ld e d a su p ern a ta n t w hich
was ev a p o ra ted on th e r o ta r y ev a p o r a to r , t o y ie l d a c le a r
y e llo w s o lu t io n o f t o t a l volum e 35 m l. Ten ml o f c o ld
4N p e r c h lo r ic a c id were added. The s o lu t io n w a s n c e n tr i-
fu g ed and th e su p ern a ta n t d ec a n ted . The p r e c ip it a t e was
23
washed w ith 28 ml o f c o ld 0 .6 I T p e r c h lo r ic a c id and
c e n tr ifu g e d . (The two su p e rn a ta n ts were p o o le d and th r e e
drops o f p h en ol re d in d ic a t o r were a d d ed . The s o lu t io n
was a llo w ed to s i t o v e r n ig h t in th e r e f r ig e r a t o r . The
su p ern a ta n t was d eca n ted from th e p r e c ip it a t e d p o ta ssiu m
p e r c h lo r a t e .
The su p ern a ta n t was p la c e d on D ow ex-l-form ate
colum n, d ia m eter 1 .3 cm, le n g th 20 cm, and washed w ith
about 600 ml o f w ater u n t i l th e e lu a te no lo n g e r co n ta in e d
u l t r a v i o l e t a b so rb in g m a te r ia l. E xtended g r a d ie n t e lu t io n
was begun w ith 2 .3 N fo rm ic a c id and a r e s e r v o ir o f one
l i t e r o f w ater s t i r r e d w ith a m agnetic s t i r r e r . T h e.
e lu ta n t was changed t o 9 -N form ic a c id a f t e r about 250 ml
and to 9 H fo rm ic a c id and 1 M ammonium f o i’mate a f t e r ap.
a d d itio n a l 600 ml o f e lu a t e were c o l l e c t e d . F in a lly th e
column was washed w ith about 50 ml o f 2 M ammonium form ate
t o remove any m a te r ia l re m a in in g . U lt r a v io le t ab sorb in g
peaks were i d e n t i f i e d by th e r a t i o s o f t h e i r ab sorb ancy
a t 280 m i^ to t h a t a t 260 mv\.
M easurements o f R a d io a c t iv it y - R a d io a c t iv it y o f s o lu t io n s
was m easured w ith a N u clea r Chicago g a s flo w c o u n te r ,
m odel D4-7. A N u clear C hicago model C100B a c tig r a p h was
,used f o r th e paper s t r i p s .
CHAPTER IV
RESULTS
Growth E xperim ents - E. c o l l K12 (\)T ~ was grown in th e
p resen ce o f v a rio u s a n a lo g s to t e s t t h e ir e f f e c t on c e l l
m u lt ip lic a t io n . T able I shows th e maximal v ia b le c e l l
count o b ta in ed in th e cou rse o f a fo u r to f i v e hour In cu
b a tio n in th e p resen ce o f v a rio u s a n a lo g s and in th e
ab sen ce o f a n a lo g . I t can be seen th a t th e p resen ce o f
AUDR, M AUDR, BUDR, and U D R r e s u lte d in th e a ttain m en t o f
a s i g n i f i c a n t l y g r e a te r number >of v ia b le c e l l s than th e
1 Hg/ml thym idine c o n t r o l,.,w h ile D M A U D R d id n ot produce
a s ig n if ic a n t in c r e a s e in v ia b le c e l l s . The r e la t iv e
maximal c e l l cou n ts a tta in e d are i l l u s t r a t e d in R igure 1 .
F igu re 2 shows th e in c r e a se in tu r b id it y o f
c u ltu r e s d uring th e cou rse o f a t y p ic a l exp erim en t,
number f i v e . F ig u re 3 shows th e changes in number o f
v ia b le c e l l s d uring th e cou rse o f experim ent 6 . From
th e se two fig u r e s i t can be seen th a t AUDR and M A U D R
c u ltu r e s f i r s t c lo s e ly p a r a lle le d th e growth o f th e 10
\^g/ml thym ine c o n tr o l, and th en d ied even more r a p id ly
than th e 1 \\g/m l c o n tr o l. There was 90-99 per cen t lo s s
o f v i a b i l i t y in two hours fo llo w in g th e a ttain m en t o f
maximal c e l l co u n t. T u rb id ity o f th e s e c u lt u r e s , however
d id not d ecrea se w ith th e d eath o f th e c e l l s . I t can
24 j
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ J
25
(T A B L E I
M AXIM A L VIABLE. C O UNT ATTAINED BY E. GOLI
K12 I" IN THE PRESENCE O P VARIOUS ANALOGS
AND MINIMAL THYMIDINE
— 7
B a c te r ia /m l x 10 f
TDR ujg/ml
Analog
1 10 1
AUDR*
1
M A U D R *
1
D M A U D R *
1
BUDR*
1
UDR:
Experim ent
1
#
4 .0 150
m m
3 ,0
2 — 420 26 16 4 .4 -
-
5
5 .2 430 28
25
4 .1 - -
4
3 .5
130 16
13
-
23
n
5 3 .3
200
15
2 i - 16
13
6
h i
190
zL zL -
18
Average 3 .8 260 21
19
3 .8 28 14
% o f c o n tr o l 100 6840 550 500 100 740 370
*£nalog c o n c e n tr a tio n s : 100 i\g/m l AUDR, 100 ug/m l M A U D R
100 Hg/ml DMAUDR, 20 i\g/m l BUDR, 100 \\g/m l U D R
/ 20 qg/m l AUDR gave 27 x 10^ b a c t ./m l; 50 Hg/ml M A U D R
gave 22 x 10^ b a c t ./m l
RELATIVE MAXIMAL CELL COUNT 1
26
68
8
6
4
2
OC
o
h I-
c r >
JL
O
O C
o
3
<
2
o
O )
O
o
OC
O
3
c r
o
3
<
2
er.
<
O
O
O C
a
3
<
< s >
*
O
O
O C
o
3
00
o
e g
oc
o
h-
«»
*
o
D
/ A
o
o
F ig . 1 - R e la tiv e M axim al_V iable Count
A tta in ed by E. d o li K12 T in th e P resen ce
o f V arious A nalogs and Minimal Thymidine
27
L U
<
O
(I )
O
O
* — l /*g. T D R / m l
j Q “ o — I O / t g T D R / m l
100/ t g A U D R / m l
o — 100 / * g M A U D R / m I
0 .
H
Q
CD
q :
3
h -
HOURS
F ig . 2 - Changes in T u r b id ity o f C u ltu res o f
E. c o l i K12 T in th e P resen ce o f AUD R or
M A U D R and Minimal Thymidine
VIABLE COUNT (L O G SCALE)
28
X — I f i t I TDR/ml
O — \ 0 fig TDR/.n!
__ 20yug TDR/m l
Q 5 0 fjug TDR/m l
HOURS i
: i
F ig . 3 - Changes i n V ia b le C e ll Count o f E. c o l i :
K12 T~ During In cu b a tio n in th e P resen ce o f AUDR
or M A U D R and Minimal Thym idine. !
29
a ls o be n o ted th a t th e t u r b id it y o f c u lt u r e s c o n ta in in g
an a lo g in c r e a s e d to a much g r e a te r e x te n t th an d id v ia b le
c e l l couhat. T h is in d ic a t e d th a t th e c e l l s were fo llo w in g
th e d e sc r ib e d co u rse fo r th y m in e le ss d e a th , e lo n g a tio n
w ith o u t c e l l d i v i s i o n . T h is was con firm ed by exam in ation
o f c e l l s w ith a m icro sco p e.
As se en in T able I , experim ent 6 , i n w hich 20 u * g
AUDR/ml were u sed in s t e a d o f 100 u^g/ml a s in p r e v io u s
ex p er im en ts, showed th a t 20 ug AUDR/ml stim u la te d growth
T - > .
t o th e same e x te n t as d id 100 u g /m l. S im ila r ly , 50 ug
MAUDR/ml seem ed to s tim u la te grow th a s w e ll a s 100 qg
MAUDR/ml.
An exp erim en t u sin g thym ine in s t e a d o f th ym id in e
t o s a t i s f y th e grow th requ irem en t gave v e r y s im ila r growth
c u r v e s . T able I I sum m arizes th e maximal c e l l co u n ts
a t ta in e d .
M A X IM A L VIABLE CO URT ATTAINED BY E. COLI
K12 T" IN THE PRESENCE O E ANALOGS A N D
MINIMAL THYM INE
B a c te r ia /m l x 10”^
Thymine tjg/ml I 10 1 1 1 1
AUDR ng/m l - 20 100 20
M A U D R ug/m l - 20 100
B a cteria /m lx lO " ^ 6 130 , 21 22 12 24
Prom t h i s t a b le i t can be se en th a t w h ile 20 qg AUDR/ml
30
and 100 i^g/ml r e s u lte d In th e same maximal v ia b le s e l l
cou n t, 20 t\g MAUDR/ml gave o n ly about one h a lf th e number
©f v ia b le c e l l s g iv e n by 100 ug MAUDR/ml. T his experim ent
a ls o dem onstrated th a t th e r e a c tio n ,
thym ine + deoxyribose-5-P O ^ ^ —— thym idine
Is not a lim it in g enzym ic r e a c tio n , s in c e thymine, in
s im ila r c o n c e n tr a tio n gave as good a growth stim u la tio n
as thym idine d id in o th er ex p erim en ts.
G-rowth exp erim en ts were a ls o c a r r ie d out w ith
£ • c o l l 15 T’ , which had been used In p rev io u s s tu d ie s
o f an alog in c o r p o r a tio n ( 1 1 ). I t can be seen from T able
I I I th a t.a n a lo g s caused about th e same e x te n t o f growth
s tim u la tio n in t h is organism as fo r K12 (A)T~. F igu re
4 shows th e tim e curve fo r c o n c e n tr a tio n o f v ia b le c e l l s
fo r experim ent 8 . Once a g a in th e I n c r e a s in g ly rap id
death o f c e l l s grown in th e p resen ce o f th e a n a lo g s Is
found.
Table IV shows a com parison o f th e e f f e c t s o f AUDR
a t c o n c e n tr a tio n s o f 10, 20, 30, and 40 i^g/ml, on th e
growth o f E. c o l l 15 T” in s y n th e tic medium w ith 1 Hg/ml
TDR.
P rep a ra tio n o f D M A from E. c o l i 15 T~- Seven l i t e r s o f
s y n th e tic c u ltu r e medium w ith about 2 xlO® b a c te r ia /m l
y ie ld e d about 6 g o f a wet w eigh t packed c e l l s . T able V I
in d ic a t e s th e t o t a l o p t ic a l d e n s ity u n its a t 260 r a i^ found
; during v a rio u s p hases o f th e p u r if ic a t io n p roced u re.
VIABLE COUNT (LO G SCALE)
31
9
8
X — I j u q T D R / m l
O — 1 0 / t g T D R / m l
& 2 0 /*g AUDR/ml
□ _ 5 0 ^tg MAUDR/ml
1 0 ®
HOURS
F ig ._ 4 - Changes in V iab le C e ll Count o f E. c o l i
15 T d u rin g In cu b a tio n i n th e P resen ce o f A U D R
or M A U D R and Minimal Thym idine.
32
TABLE I I I
M A XIM A L VIABLE CO UNT ATTAINED BY E. COLI
15 T“ IN THE PRESENCE O E ANALOGS AND MINIMAL THYMIDINE
•’ j n
B a c te r ia /m l x 10 '
TDR qg/m l
I 10 1 1
Analog
AUD R M A U D R
Experim ent #
7
7 .6
333
—
3 5 .5 a
8
1 1 .7
86 18b
1 3 .3 °
9 8 .5 105 2 0 .4
-
10
- 2 §
2 1 .3 d
-
Average
9 .3
161 20
% o f c o n tr o l 100
........1 7 3 P ..............
215
a , 100 ug MAUDR/ml; to, 20 ug AUDR/ml; c , 50 l^g MAUDR/ml;
d , 30 ug AUDR/ml
TABLE IV
M AXIM A L VIABLE CO UNT ATTAINED BY E. COLI
15 T“ IN THE PRESENCE O P VARIOUS CONCENTRATIONS O P AUDR
B a c te r ia /m l x 10"" ^
Hg/ml AUDR E x p t. 10 E xp t. 11
10 7.9
20 1 0 .9 1 3 .1
30 2 1 .3 1 6 .3
40 1 8 .4
33
TABLE V
PURIFICATION O F BRA FR O M E: COLI 15 T '
S tep t o t a l O B
260
E x tr a c tio n o f n u c le ic a c id s from
ly s e d c e l l s 1130
Treatm ent w ith RHAase
Treatm ent w ith tr y p s in
54-0
34-5
P r e c ip it a t io n w ith is o p r o p y l a lc o h o l 80
F o llo w in g trea tm en t w ith RNAase th e BRA p r e p a r a tio n
appeared t o be f r a c t io n a te d and no lo n g e r h ig h ly
p o ly m e riz ed .
A n a ly sis o f N u c le o tid e s from th e H ydrolyzed BRA.
P r e p a r a tio n - F igu re 5 shows th e r e s u l t s o f an exten d ed
g r a d ie n t e lu t io n o f th e BRA h y d r o ly sa te from a Bow ex-1-
form ate colum n. I t can be se en t h a t th e n u c le o t id e s were
se p a r a te d in t o fo u r p ea k s.
The f i r s t peak i s w e ll se p a r a te d from th e o th e r s
and i s i d e n t i f i e d by th e r a t io o f o p t ic a l d e n s it y a t 280mn
and 260 mi\ to be d e o x y c y tid in e m onophosphate (dCMP).
T h is peak c o n ta in e d a t o t a l o f 1 0 .0 6 O B u n it s a t 280 m u *,
th e in a c id fo r dCMP. From th e m olar a b so r b tio n
I u k aJ L
c o e f f i c i e n t o f dCM P i t i s p o s s ib le to c a lc u la t e th e
number o f m icrom oles o f dCM P w hich were r e c o v e r e d .
34
6 2 so = ^-5»2 x 10^ OD/ml. T h erefore i t can be c a lc u la te d ,
th a t th e r e was a t o t a l o f 0 .7 6 5 nm oles dG M P re co v e red
from th e h y d r o ly s a te .
The second p ea k , w ith a 2 8 0 /2 6 0 r a t io n o f 0 .5 6 ,
r e p r e s e n ts th e c a r r ie r am inou ridin e - 5 '-m onophosphate
w hich was added to th e h y d r o ly s a te . The peak o f r a d io
a c t i v i t y i s seen to co rresp o n d to t h i s peak o f u l t r a v i o le t
ab so rb in g m a te r ia l. The t o t a l number o f co u n ts d e te c te d
in t h i s peak was 4283 • The r e c o v e r y o f t h i s amount o f
r a d io a c t iv i t y in t h i s peak r e p r e s e n ts in c o r p o r a te d AUDR,
is o l a t e d a s th e m onophosphate.
I t can be se e n t h a t th e t h ir d peak i s n o t e n t i r e l y
d i s t i n c t from th e fo u r th p eak . These two peaks r e p r e se n t
in c o m p le te ly se p a r a te d th ym id in e m onophosphate and d eoxy-
gu a n id in e m onophosphate. The r a t io o f a b so rb a n c ies a t
260 and 280 m u, fo r th e two peaks were 0 .7 5 and 0 .6 4
r e s p e c t iv e ly .
A n a ly s is o f A cid S o lu b le F r a c tio n o f t h e . C e ll L y sa te -
Extended g r a d ie n t e lu t io n o f th e p e r c h lo r ic a c id s o lu b le
f r a c t io n o f th e o r ig in a l c e l l ly s a t e gave numerous i l l -
d e fin e d p e a k s, s e v e r a l o f w hich co n ta in e d sm all b u t *
s i g n i f i c a n t amounts o f r a d io a c t iv i t y . T h is r e s u lt in d ic a
te d th a t th e r e was o b v io u s ly no accu m u la tio n o f th e m ono-,
d i - , and tr i-p h o s p h a te s o f AUDR i n th e a c id s o lu b le
n u c le o tid e p o o l o f th e b a c t e r ia .
35
^ 8
0
< 0
a
1 6
> -
u
<4
c o
o c
22
ca
« *
120
2 S f i j a e = o 7 2
2 6 0 " !»
80
40
160 120 80 40
TUBE N U M B E R
F ig . 5 - G radient E lu tio n o f a. H yd rolysate,
o f D K A from 'E- c o l l 15"T“ Grown in th e
P resen ce ©f C-AUDR and Minimal Thyx&idine.
CHAPTER V
DISCUSSION
I t was se e n t h a t AUDR a t c o n c e n tr a tio n s o f from
20 qg to 100 u g /m l, and. M A U D R a t c o n c e n tr a tio n s o f from
30 to 100 n g /m l, p e r m itte d c u lt u r e s o f s t r a in K12 (x ) T
t o a t t a in c o n c e n tr a tio n o f v ia b le c e l l s f i v e tim e s a s h igh
a s th e 1 mg/ml TDR c o n t r o l. In s t r a in 15 T~, AUDR a t
c o n c e n tr a tio n s o f from 20 mg/ml to 50 qg/m l p erm itte d
c u lt u r e s to rea ch a c o n c e n tr a tio n o f v ia b le c e l l s a l i t t l e
more th a n tw ic e a s h ig h a s th e 1 qg/m l TDR c o n t r o l. In
t h i s s t r a in th e 1 i\g TDR/ml c o n tr o l a t ta in e d a h ig h e r
maximal c e l l count b e fo r e d y in g .
One e x p la n a tio n o f t h i s ob served enhancement o f
grow th and l a t e r r a p id d ea th i s th a t th e a n a lo g s may
s u b s t it u t e f o r thym ine to p erm it s y n t h e s is o f new DNA and
c e l l d i v i s i o n . More in c o r p o r a tio n and an in c r e a s in g amount
o f a n a lo g i n th e DNA m ight r e s u lt i n DNA w hich d o es n ot
fu n c tio n n o rm a lly , i . e . ; w hich can n ot r e p l ic a t e a n d /o r
produce "good" DNA. T h is would accou n t f o r th e more
r a p id l o s s o f v i a b i l i t y f o r c u lt u r e s grown in th e p resen ce
o f AUDR or M A U D R th a n f o r th e 1 qg TDR/ml c o n t r o l.
A nother p o s s ib le e x p la n a tio n fo r th e enhancement
o f grow th i s th a t th e p r e se n c e o f th e a n a lo g i n th e culture
medium may e x e r t a sp a r in g e f f e c t on th e sm a ll amount o f
TDR w hich i s i n th e medium, by com peting fo r enzymes w hich
break down TDR. T h is would a llo w th e c e l l to u t i l i z e more
TDR, te m p o r a r ily p o stp o n in g th e o ccu rren ce o f th y m in e le ss
d e a th . I t was d e c id e d t o t e s t t h i s p o s s i b i l i t y by u sin g
UDR i n c o n c e n tr a tio n s e q u iv a le n t t o th e c o n c e n tr a tio n s o f
an alog u sed . I t was th ou gh t th a t UD R would n ot be in c o r
p o ra ted i n s i g n i f i c a n t amounts and would th u s r e p r e s e n t a
c o n tr o l on th e n o n - s p e c if ic e f f e c t s o f p y r im id in e s. I t
was found th a t UD R d id b r in g about an enhancement o f
grow th, a s se e n in T able I . However, Cohen e t a l . , (6 3 )
14-
r e p o r t ed t h a t r a d io a c t iv i t y from u r a c il - 2 - C was
re c o v e re d from th e D H A o f E. c o l i 15 T ^ 'U - grown fo r a s
sh o rt a tim e a s 10 m in u tes i n a medium supplem ented w ith
14-
2 qg th y m id in e/m l and 10 ug/m l 2 - C u r a c i l . Thus some
o f th e enhancem ent o f grow th by UDR m ight a ls o be e x p la in
ed by th e in c o r p o r a tio n o f u r a c il in t o th e D H A in p la c e o f
th ym in e. In any c a s e , AUDR and M A U D R c o n s i s t e n t l y a llo w ed
a h ig h e r l e v e l o f grow th th an d id UDR. Thus even i f much
o f th e U D R s t im u la t io n o f grow th was due to a sp a rin g
e f f e c t AUDR and M A U D R m ight have an e f f e c t over and above
t h i s , s u g g e s tin g th a t th e y were in c o r p o r a te d in t o th e DHA.
A n a ly sis o f th e exp erim en t d e sig n e d to t e s t t h i s
p o s s i b i l i t y fo r AUDR shows d e f i n i t e l y th a t AUD R was in c o r
p o r a te d , s in c e a s i g n i f i c a n t amount o f r a d io a c t iv i t y was j
r e co v e r ed as th e m onophosphate from th e D H A p r e p a r a tio n .
There i s some d i f f i c u l t y in q u a n t it a t iv e ly e s tim a tin g th e
38
e x te n t o f AUDR in c o r p o r a tio n from th e d a ta , s in c e y i e l d s
o f n u c le o t id e s were so low .
The f i n a l y i e ld o f p u r if ie d D H A o b ta in ed from
seven l i t e r s o f c e l l s was v e r y sm a ll— about 3 -4 mg ou t of
a t o t a l o f about 24 mg in th e c e l l s . As n o ted t h i s D H A
appeared to be fr a c t io n a te d and n ot h ig h ly p o ly m eriz ed .
Dunn and Sm ith (3 0 ) r e p o r te d th a t th e DH A from E, c o l i
15 T~ grown i n lim it in g amounts o f th ym id in e i s more
e a s i l y f r a c t io n a te d th a n normal DHA. S in ce a m in o u ra cil
c a u se s an in c r e a s e in 6-m eth ylam inop u rin e co n te n t o f D H A
from E. c o l i 15 (4 8 ) i t i s p o s s ib le th a t AUDR m ight
have a s im ila r e f f e c t . D H A w ith a h ig h c o n te n t o f
6-m eth ylam inop u rin e i s b e lie v e d t o have abnormal b a se
r a t i o s and perhaps to be n o n -fu n c tio n a l ( 2 ,4 9 ) . I t i s
th u s p ro b a b ly f r a g i l e and t h i s would e x p la in why our
e x t e n s iv e ly p u r if ie d p r e p a r a tio n was f r a c t io n a te d and in
such low y i e l d .
The y i e l d o f h y d ro ly sed n u c le o t id e s was a ls o v e r y
low . Erom e ig h t y o p t ic a l d e n s it y u n it s o f D H A we sh o u ld
have ex p ec te d to re co v e r more th an e ig h t y o p t ic a l d e n s it y
u n it s o f n u c le o t id e s , i f h y d r o ly s is were co m p lete.
However, o n ly 10 o p l i c a l d e n s it y u n it s o f dCM P were
i
r e c o v e r e d .
Table VI shows th e b a se co m p o sitio n o f E. c o l i
15 T~ grown under v a r io u s grow th c o n d it io n s , a cc o rd in g to
Dunn and Sm ith ( 4 8 ) .______________________________________ ______
39
TABLE VI
PROPORTIOE O P BASES IE E. COLI 15 T~ DEA
( MOLES/lOO M OLES TOTAL BASES RECOVERED)
B a c te r ia l P re p a r a tio n A G - C T 6 -M eth y l-
am inopurine
c o n tr o l 2 5 .6
2 7 .5
2 6 .1 2 2 .4 0 .4 2
5-AU in h ib it e d 2 2 .4 2 7 .8
2 6 .7
1 9 .1 4 .1
•^hymine d e f i c i e n t 2 2 .0 2 8 .6
2 7 .5 1 8 .9 3 .1
I t can be se en th a t th e p r o p o r tio n o f c y t o s in e in
th e DEA i s r e l a t i v e l y c o n sta n t under d if f e r e n t grow th
c o n d it io n s . The y i e l d o f dCMP can th u s be used t o c a lc u
l a t e th e amount o f DEA w hich was h y d r o ly z e d , or th e t o t a l
number o f u m oles o f n u c le o t id e s r e le a s e d . Prom th e €
max
o f dCM P th e y ie ld o f dCMP was c a lc u la te d t o be 0 .7 6 5 umoles
Taking dCM P to be e q u iv a le n t to 2 6 .7 p er c e n t o f th e t o t a l
n u c le o t id e s , i t was c a lc u la t e d th a t th e r e was a t o t a l o f
2 .8 6 5 H m oles o f n u c le o t id e s h y d ro ly ze d .
On t h i s b a s is we can c a lc u la t e th e p er c e n t o f the
t o t a l b a se s r e p r e se n te d by th e r a d io a c tiv e AUD R monophos-%
p h ate r e c o v e r e d . Prom th e known s p e c i f i c a c t i v i t y o f th e
14
2 - C AUDR u se d , we can c a lc u la t e th e number o f \\ m oles
r e c o v e r e d . Such a c a lc u la t io n y i e l d s th e in fo r m a tio n th a t
0 .0 4 9 q m oles o f AUDR m onophosphate were r e c o v e r e d . T h is
amount co rresp o n d s to AUDR in c o r p o r a tio n o f 1 .7 1 p er c e n t.
T h is fig u r e i s c a lc u la t e d u sin g th e knowledge th a t thym ine
40
n orm ally r e p r e s e n ts 2 2 .4 p er c e n t o f th e b a s e s .
U n fo r tu n a te ly , th e s e p a r a tio n o f T M P and dG M B
by exten d ed g r a d ie n t e lu t io n from D ow ex-l-form ate was n o t
com p lete and i t was n ot p o s s ib le t o c a lc u la t e th e amounts
o f th e s e two n u c le o t id e s . N e ith e r was th e y i e ld o f
n u c le o t id e s la r g e enough t o a llo w th e d e t e c t io n o f
6-m eth ylam inop u rin e in th e DNA.
Dunn and Sm ith were unable t o d e t e c t any amino
u r a c il in DN A o f E. c o l i 15 T~ grown in th e p resen ce o f
135 ami no u r a c il/m l and 2 .5 u » g thym in e/m l ( 4 8 ) . T h eir
chrom atographic methods were s e n s i t i v e enough t o d e t e c t
0 .4 2 p er c e n t o f . th e t o t a l b a se s a s 6-m eth ylam in op u rin e.
I t i s o f i n t e r e s t th e n to ask w hether th e la c k o f in c o r
p o r a tio n o f a m in o u ra cil can be e x p la in e d by th e i n a b i l i t y
o f th e b a c t e r ia to co n v ert a m in o u ra cil t o th e d eoxyriboside
Our d a ta have shown th a t th e d e o x y r ib o sid e can be in c o r
p o ra ted in amounts w hich m ight have b een d e te c ta b le by
t h e ir a n a ly t ic a l te c h n iq u e . Thus u n le s s p e r m e a b ility and
r a t e s o f d eg r a d a tio n are s i g n i f i c a n t l y d if f e r e n t f o r th e
f r e e b ase and th e n u c le o s id e , th e e v id e n c e m ight s u g g e s t
th a t E. c o l i 15 T~ i s unable to co n v ert a m in o u ra cil to th e
d e o x y r ib o s id e , a lth o u g h presum ably i t i s a b le to co n v er t
th e d e o x y r ib o sid e to th e tr ip h o s p h a te l e v e l s in c e th e
n u c le o s id e was in c o r p o r a te d . W e know from th e work o f
( t
Lehman e t a l . , (6 5 ) th a t DN A p olym erase r e q u ir e s th e t r i
p h o sp h a tes o f th e b a se s f o r DNA s y n t h e s is . Thus i f an
a n a lo g i s in c o r p o r a te d we know th a t i t must go through
th e pathway d e o x y r ib o sid e .— hi m o n o p h o s p h a t e d ip h o s p h a t e
— 4 tr ip h o s p h a te .
I t i s o f i n t e r e s t th a t M A U D E (b u t n o t DM AUDE)
behaved in a manner a lm o st i d e n t i c a l to AUDR in a l l
grow th ex p er im en ts, e x c e p t th a t h ig h e r c o n c e n tr a tio n s
seem ed to be req u ired to b r in g about a tta in m en t o f s im ila r
c o n c e n tr a tio n s o f v ia b le c e l l s . T h is may in d ic a t e th a t
M A U D R i s a ls o in c o r p o r a te d in t o DNA in t h i s organ ism ,
w h ile D M A U D R i s n o t. I f t h i s were so i t would seem t o
in d ic a t e th a t th e s iz e o f th e group in th e f i v e p o s it io n
i s in d eed n ot v e r y im p o r ta n t, a s su g g e ste d by Dunn and
Sm ith ( 3 0 ) , who found t h a t C1U was in c o r p o r a te d to a
g r e a te r e x te n t th a n BU in E. c o l i 15 T~ DNA, even though
th e r a d iu s o f th e bromo group i s more s im ila r to th e
m ethyl group o f thym ine th an th e e h lo r o group i s . The
m ethylam ino and dim ethylam ino groups b o th have a van der
W aal's r a d iu s o f 3*2A (6 4 ) compared to 2.0A fo r th e m ethyl
group o f th y m in e, y e t o n ly M A U D R s tim u la te s grow th. The
amino grou p , on th e o th e r hand, has a r a d iu s i d e n t i c a l to
t h a t o f th e m ethyl group and y e t we have se e n t h a t i t i s
in c o r p o r a te d to a l e s s e r e x te n t th an any o f th e h alogenated.
u r a c i l s . Dunn and Sm ith re p o r te d (3 0 ) t h a t C1U r e p la c e s
64 p er c e n t o f th y m in e, BU r e p la c e s 31 p er c e n t o f th e
thym ine and IU , 29 p er c e n t, under c o n d itio n s s im ila r to
th o se i n our ex p er im en ts. I t seem s c le a r th a t o th e r_______
p r o p e r tie s o f th e group i n th e f i v e p o s it io n must govern
e x te n t o f in c o r p o r a tio n .
CHAPTER VI
SU M M A R Y
A m inodeoxyuridine (AUDR) and m ethylam inode o x y u r i-
d in e (MAUDR) enhance grow th i n two th y m in e le s s s t r a in s o f
E. c o l i . C u ltu r e s grown i n th e p re sen ce o f th e s e a n a lo g s
and a lim it in g amount o f th ym id in e a t t a in a c o n c e n tr a tio n
o f v ia b le c e l l s w hich i s h ig h e r th a n t h a t a t ta in e d by a
c o n tr o l c u ltu r e grown i n lim it in g th ym id in e a lo n e .
C u ltu res grown in th e p r e se n c e o f th e s e a n a lo g s were a ls o
o b served to have a more r a p id l o s s o f v i a b i l i t y th a n th o s e
grown i n lim it in g thym id ine a lo n e . These o b s e r v a tio n s
in d ic a te d th a t AUD R and M A U D R m ight be in c o r p o r a te d in t o
th e DNA o f th e s e b a c t e r ia l s t r a in s .
In ord er to t e s t t h i s p o s s i b i l i t y , E. c o l i 15 T“
14
was grown in th e p resen ce o f C-AUDR and a lim it in g
amount o f th y m id in e. DH A from b a c t e r ia grown in su ch a
medium was i s o l a t e d , p u r if ie d and h y d ro ly zed e n z y m a tic a lly
t o n u c le o t id e s . . A m in o u rid in e-5 ' -m onophosphate was added
t o th e h y d r o ly sa te a s a c a r r ie r f o r any c L A M P w hich m ight
have been p r e s e n t i n th e h y d r o ly s a te . The h y d r o ly sa te
was se p a r a te d in t o component n u c le o tid e f r a c t io n s by g ra d -
jie n t e lu t io n from a column o f D o w ex -l-fo rm a te. Pour peaks
|
jwere o b serv ed , co rresp o n d in g t o dCMP, c a r r ie r AUMP, dGMP,
and TMP, id e n t if ie d , by t h e i r r a t io s o f absorbancy a t 280
mi\ t o absorbancy a t 260 m t^. A s in g le r a d io a c tiv e peak
___________________________________43 ______________
44
was found to corresp on d e x a c t ly to th e u l t r a v i o l e t peak
i d e n t i f i e d a s c a r r ie r A UK S?, in d ic a t in g t h a t th e r a d io -
14
a c t i v i t y was a s s o c ia t e d w ith G-dAUMF w hich i s e lu te d
sim u lta n e o u sly w ith A U M P from th e Ion exchange r e s in . 0?he
p r esen c e o f dAU&P in th e h y d r o ly sed D M A i s im p ortan t
14
ev id e n c e th a t C-AUDR was in c o r p o r a te d in t o th e DRA,
presum ably v i a p r io r tr a n sfo r m a tio n t o th e m ono-, di-1 -,
and tr i-p h o s p h a te . A q u a n tita t iv e e s tim a tio n o f th e extent
o f AUDR in c o r p o r a tio n was made. AUDR was c a lc u la te d t o he
in c o r p o r a te d to th e e x te n t o f 1 .7 p er c e n t o f th e t o t a l
b a se s or 7*6 p er c e n t rep lacem en t o f thym ine.
BIBLIOGRAPHY
1 . C h a r g a ff, E. , "The ch em ica l B a s is o f H ered ity" ed . ,
M cElroy, W . D ., and G la s s , D. , Johns H opkins
U n iv e r s ity P r e s s , 521 (1 9 5 7 )
2 . C ric k , P. H. C. , "The Chem ical B a s is o f H ered ity"
e d . , M cElroy, W . D. , and G la s s , D. , Johns Hopkins
U n iv e r s ity P r e s s , 532 (1957)
3 . A very, 0 . T. , MacLeod, C. M ., and McCarty, M .
J . E x p tl. Med. 7 9 , 137 (1 9 4 4 )
4 . B e a d le , G. W., P h y s io l. R e v ., 2 £ , 643 (1 9 4 5 )
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Asset Metadata
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Kabat, Susan Frances (author)
Core Title
Studies of aminodeoxyuridine, methylaminodeoxyuridine and dimethylaminodeoxyuridine
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Master of Science
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Biochemistry and Nutrition
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Kabat, Susan Frances
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University of Southern California Dissertations and Theses