536 P. BOULANGER, R. OSTEUX, J. BERTRAND VOL.

2 9 (1958)
Rt~SUMt~

La L-aminoacide-d6shydrog6nase du foie de D i n d o n (Meleagris gallopavo L.) d6samine in vitro la

5-hydroxylysine en un p r o d u i t r6ductible en acide 5-hydroxypip6colique.

SUMMARY

The L-aminoacid dehydrogenase p r e p a r e d from t u r k e y liver deaminates 5-hydroxylysine in vitro

to f o r m a p r o d u c t t h a t can be reduced catalytically to 5-hydroxypipecolic acid.

BIBLIOGRAPHIE

1 A. I. VIRTANEN ET S. KARI,Acta Chem. Scand., 8 (1954) 129o; 9 (1955) 17o.

2 N. GROBBELAAR, J. K. POLLARD ET F. C. STEWARD, Nature, 175 (1955) 703 •
3 p. BOULANGER ET R. OSTEOX, Biochim. Biophys. Acta, 21 (1956) 552; 26 (1957) 143.
4 D. D. VAN SLYKE ET A. HILLER, Proc. Natl. Acad. Sci. U.S., 7 (1921) 185.
S. B. SCHRYVER,H. V. BUSTON ET D. H. MUKHERJEE, Proc. Roy. Soc. (London), 9 8 B (1925) 58.
6 D. D. VAN SLYKE et al., Proc. Soc. Exptl. Biol. Med., 38 (1938) 548 ; J. Biol. Chem., 133 (194 o) 287 ;
143 (1942 ) 433-
7 p. DESNUELLE ET S. ANTONIN, Biochim. Biophys. Acta, I (1947) 5 o.
s j. C. S~EEHAS ET W. A. BOLHOFER, J. Am. Chem. Soc., 72 (195 o) 2466.
9 A. H. GORDON, Biochem. J., 45 (1949) 99.
10 T. ASTRUP, G. CARLSTROM ETA. STAGE, Acta Physiol. Scand., 24 (1951) 2o2.
11 M. BARBIER E T E . LEDERER, Biochim. Biophys. Acta, 8 (1953) 590.
12 G. VAN ZYL, E. E. TAMELEN ET G. D. ZUIDEMA, J. Am. Chem. Soc., 73 (1951) 1765-
13 W. S. FONES, J. Am. Chem. Soc., 75 (1953) 4865 .

Re~u le 20 f6vrier 1958

P R O P E R T I E S O F R I B O N U C L E I C ACID T U R N O V E R IN T 2 - I N F E C T E D
ESCHERICHIA COLI

L. A S T R A C H A N AND E. V O L K I N
Biology Division, Oak Ridge National Laboratory,* Oak Ridge, Tennessee (U.S.A.)

Although infection of Escherichia coli with bacteriophage 1"2 immediately prevents

any further net synthesis or loss of host RNA 1, some metabolic activity of the RNA
(ribonucleic acid) can be detected by isotope-tracer techniques 2. With the use of 32p_
labeled orthophosphate, turnover of RNA phosphorus is readily demonstrated 3. It
was also noted that, shortly after addition of 32p0 4, the amount of isotope incorporated
into RNA is greater than that found in DNA, although DNA eventually incorporates
far more isotope than does RNA because of the extensive net synthesis of DNA in
this system. Furthermore, nonuniform activity of RNA in phage-infected cells is
revealed by the finding that the alkali-produced RNA mononucleotides do not have
equal specific radioactivities. These data suggested that bacteriophage infection
induces the bacteria to synthesize a new kind of RNA, the base composition of which
might be that obtained from the percentage of total RNA radioactivity associated
* Operated b y Union Carbide Corporation for the U. S. Atomic E n e r g y Commission.
Re/erences p. 544.
voL. 2 9 (1958) RIBONUCLEIC ACID TURNOVER IN E. coli 537

with each of the mononucleotides; i.e., 18 % cytidylic, 30 % adenylic, 30 % uridylic,

and 22 % guanylic acids. These proportions are quite different from the chemical
composition of E. coli RNA, which yields 23 % cytidylic, 24 % adenylic, 22 % uridylic,
and 31% guanylic acids. It is interesting to note that the analogous deoxyribonu-
cleotides of T2 bacteriophage DNA have relative proportions 4 similar to those just
described for the radioactive RNA of bacteriophage-infected bacteria.
The possibility remained, however, that the nonuniform specific activities of the
RNA mononucleotides were attributable to experimental conditions not related to
phage infection. Experiments are presented here which compare infected and un-
infected cells with regard to the distribution of radioactivity in the RNA mono-
nucleotides after short-term incubation in the presence of 32P04. In addition, this
report relates the effects of altered experimental conditions on the turnover of RNA
and the synthesis of DNA. The modifications used were: (a) RNA turnover was
measured at different times after infection and (b) parallel experiments were per-
formed in broth and synthetic medium to evaluate the effects of the culture medium ;
and (c) most of the labeled acid-soluble material was washed out of the cells after
the period of isotope incorporation, resulting in a quantitative relation between
release of ~2p from RNA and accumulation of 3,p by DNA.

MATERIALS AND METHODS

Peptone broth was t h a t of HERSHEY AND CHASE5.

Dephosphorylated peptone broth w a s p r e p a r e d by dissolving t h e ingredients for i 1 of p e p t o n e
b r o t h in 80o ml of w a t e r and t h e n adding 2.5 ml of I M MgC18 followed b y the dropwise, w i t h
stirring, addition of 5 ml of concentrated NH4OH. The m i x t u r e w a s chilled for t w o h o u r s in a
refrigerator. After centrifugation to r e m o v e the precipitate, 6.2 g of t r i s ( h y d r o x y m e t h y l ) a m i n o -
m e t h a n e w a s added, the p H a d j u s t e d to 8.o with N HC1, and t h e v o l u m e m a d e to I 1. This resulted
in a reduction of the concentration of inorganic p h o s p h a t e f r o m 8o~9o # g / m l to 1-7 #g/ml.
The previously described synthetic medium 8 was modified to include FeC13 at a final con-
centration of IO -5 M. Some of the c o n s t i t u e n t s for this m e d i u m were suggested by MANSONe.
Adsorption medium had final concentrations of o.I M NaCI, o.ooi M MgC18, o.oooi M CaC18,
and o.oi M t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e at p H 7.7.

Conditions ot infection
O v e r n i g h t - g r o w n cells of E. coli B were diluted to a titer of 2- 4" IO7 cells/ml and t h e n in-
c u b a t e d w i t h aeration at 37 ° until a titer of 3 - 5 ' lO8 cells/ml was obtained. I n m o s t experiments,
these log-phase cells were t h e n directly infected in the g r o w t h m e d i u m w i t h a IO- to IS-fold
multiplicity (phage/bacteria) of T 2 r + bacteriophage 7 (zero time). I n one e x p e r i m e n t (Fig. 2),
the bacteria were centrifuged, w a s h e d with cold adsorption m e d i u m , resuspended in adsorption
m e d i u m at 37 ° and were t h e n infected. After 5 m i n of aeration at 37 ° the bacteria were p u t into
p e p t o n e b r o t h g r o w t h m e d i u m (zero time). The phage-infected bacteria were always aerated and
incubated at 37 ° . Assays for free phage, total plaque formers, uninfected and infected bacteria,
performed as described b y AI)AMSs, indicated t h a t in all e x p e r i m e n t s at the time of isotope ad-
dition, the n u m b e r of uninfected bacteria was Io -8 to lO 4 of the n u m b e r of infected bacteria.

Experimental procedure
38p, obtained f r o m the Oak Ridge National L a b o r a t o r y in the form of inorganic p h o s p h a t e ,
was neutralized and added to the culture of phage-infected bacteria at various times after in-
fection. 4 to I o min after 38p addition, t h e p h o s p h o r u s specific activity was reduced by increasing
the 31p o r t h o p h o s p h a t e concentration Ioo- to 3oo-fold. I n one e x p e r i m e n t (Fig. 5), after 4 min
for isotope incorporation, 2 1 of the infected cells were quickly chilled by the rapid addition of I 1
of crushed ice containing o.i mole of NaC1 and o.oi mole of sodium p h o s p h a t e at p H 7.5. After
centrifugation at 15oo x g in the cold, the cells were washed w i t h I5OO ml of s a l i n e - p h o s p h a t e
(above concentration), recentrifuged, and suspended in 200 ml of cold s a l i n e - p h o s p h a t e . 2 1 of
d e p h o s p h o r y l a t e d broth, made o.oi M w i t h respect to o r t h o p h o s p h a t e , were w a r m e d to 37 ° and
t h e n mixed with the cold suspension of infected bacteria. Aeration and incubation at 37 ° were
resumed.
Relerences p. 544.
538 L. ASTRACHAN, E. VOLKIN VOL. 29 (1958)

Chemical techniques
P o r t i o n s of t h e labeled, i n f e c t e d b a c t e r i a l c u l t u r e w e re chilled in a n ice b a t h a n d simul-
t a n e o u s l y acidified w i t h c o n c e n t r a t e d p e r c h l o r i c acid to a final c o n c e n t r a t i o n of 0. 5 N acid.
Nucleic acids a n d o t h e r c h e m i c a l f r a c t i o n s w e r e o b t a i n e d b y a p p l y i n g t h e m e t h o d of TYNER et al. 9
w i t h s o m e m i n o r m o d i f i c a t i o n s2. The i o n - e x c h a n g e s e p a r a t i o n of t h e a l k a l i - p r o d u c e d R N A mono-
n u c l e o t i d e s from each o t h e r a n d from D N A has been d e s c r i b e d 2. A t t i m e s , t h e i o n - e x c h a n g e
p r o c e d u r e w a s modified so t h a t t h e R N A m o n o n u c l e o t i d e s w e re n o t i n i t i a l l y s e p a r a t e d from each
o t h e r b u t were collected t o g e t h e r b y e l u t i n g w i t h 60o m l of o.oi N HC1. T o t a l R N A so o b t a i n e d
w a s i m m e d i a t e l y n e u t r a l i z e d w i t h a s l i g h t excess of N H a O H and, in a d d i t i o n to o t h e r a na l ys e s , w a s
a s s a y e d for t h e p r e s e n c e of a n y r a d i o a c t i v i t y a s s o c i a t e d w i t h i n o r g a n i c p h o s p h a t e TM. I n o r g a n i c
a n d t o t a l p h o s p h o r u s w e r e d e t e r m i n e d b y t h e m e t h o d of GRISWOLD et al. n . U l t r a v i o l e t l i g h t ab-
s o r p t i o n w a s m e a s u r e d in a B e c k m a n DU s p e c t r o p h o t o m e t e r , a n d r a d i o a c t i v i t y m e a s u r e m e n t s
were p e r f o r m e d as p r e v i o u s l y d e s c r i b e d 2.

RESULTS

The turnover of RNA in phage-infected bacteria was measured previously in experi-

ments where 32p-labeled orthophosphate was added to the medium 3 to 5 min after
infection 3. This procedure satisfied the requirement that complete phage adsorption
occur before isotope addition and provided information about some metabolic events
occurring soon after infection. Since it is known, however, that abrupt changes in the
rate of synthesis of DNA occur 8 to I0 rain after infection 1, it became of interest
to measure the turnover of RNA at various times after infection.
At various times after infection, isotope was presented to cells of E. coli that had
been cultured and infected in dephosphorylated broth. Five minutes after 32p was
added, the isotope was virtually diluted out by the addition of unlabeled orthophos-
phate buffer in i4o-fold excess. Fig. i summarizes the changes in 32p content of RNA
and DNA for the various parallel experiments. (It should be noted that the RNA
scale is magnified threefold with respect to the DNA scale.) It can be seen that the
turnover curves for RNA change markedly with the passage of time after infection.
Thus the amount of isotope incorporated into RNA during the 5-min period starting
I min after infection is greater than the amounts incorporated in any subsequent
5-min period. The extent of incorporation decreases for tile first 16 min after infection,
after which it maintains a relatively steady value. Similarly, in the experiments
started at the earlier times after infection, the rates at which isotope leaves RNA are
greater than at the later times. While the rate of RNA turnover decreases with time
after infection, the rate of 32p incorporation into DNA increases. A result of these
changing rates is that if isotope is added I i or more minutes after infection, more 32p
promptly appears in DNA than in RNA, the reverse of the situation found when
isotope is added earlier.
Although this demonstrates that the turnover of RNA changes with time, the
possibility exists that the very high incorporation observed shortly after infection is not
a property of phage-infected cells but is an indication of participation either b y un-
infected cells or by infected cells that still retain some RNA synthesizing property of
uninfected cells.
Ultraviolet absorbancies and some confirmatory total phosphorus assays of
isolated mononucleotides reveal that the RNA base composition is the same for
infected and uninfected cells, the average composition being 23 % cytidylic, 24 %
adenylic, 22 % uridylic, and 31% guanylic. There is, however, a marked qualitative
difference in the mode of incorporation of isotope into the RNA of infected and
Re]erences p. 544.
v o L 29 (~958) RIBONUCLEIC ACID TURNOVER IN E. coli 539

~2 4
A .11 ( B
41
•

ONA ~0~
.,~
-~ ~ ~g
7~5 z~
-6~ ~.-
. so
c-
4o
~_
~
r\/,, 6~
s~
>-2~
c-
6~

o
i

(3 20 3b 40 50 ~o 6o 0

12 ~2 p3,
TIME(re,n)
T~
t
12 p3, TIME {mml
EXCESS EXCESS EXCESS
4 12

/
H
io~ ~o~

!' / =
9~
.= o

/
6~
e:

Lz

I
I
8~ z,a
~c

>

c~
/
e, ; IV
£
/
o ,o so ,;b sb 60 ~ 6o ~0 " " 4b so 6o
! I I TIME{m,~) 'b ~ TTIME(m,n) 5b
T2 p3Z p31 p
EXCESS EXCESS EXCESS

Fig. I. Turnover of RNA-32P and accumulation of DNA-S2P as a function of time after infection
of isotope addition (dephosphorylated broth). To parallel samples of infected cells, a2P-labeled
orthophosphate was added at designated times after infection. Five minutes after ~2p was added,
its specific radioactivity was reduced by adding sufficient pH 7.5 phosphate buffer (alp) to increase
the orthophosphate concentration from 1.4 to 1.8 #g of P/ml to 200 to 250 #g of P/ml. To correct
for variations in cell numbers, the values for 32p in RNA and DNA were adjusted to what would
be found in a sample containing iooo optical density units of RNA (26o m#). The scale for RNA
is magnified threefold relative to the DNA scale.

uninfected cells. Since the isotope d i s t r i b u t i o n a m o n g the mononucleotides from u n -

infected cells (Table I) is the same as the R N A base composition, these m o n o n u c l e o -
tides obviously have equal specific radioactivity. (The slight differences found between
actual base composition a n d isotope d i s t r i b u t i o n were a t t r i b u t a b l e to e x p e r i m e n t a l
variations in the a m o u n t s isolated a n d not to changes in specific activity.) W i t h
infected cells, on the other hand, decidedly n o n u n i f o r m specific activities were observed,
especially where 32p was incorporated I to 4 m i n after infection. This early rearrange-
m e n t of the p a t t e r n of isotope incorporation indicates t h a t the high incorporation of
3~p into R N A shortly after infection is not complicated b y an R N A - s y n t h e s i z i n g
p r o p e r t y retained from uninfected cells. After isotope dilution, when isotope is lost
from RNA, the R N A mononucleotides gradually approach uniform specific radio-
a c t i v i t y at a rate d e p e n d e n t on the rate of 32p loss. I n most experiments, n e a r l y
u n i f o r m specific radioactivities were e v e n t u a l l y a t t a i n e d ; b u t when isotope was a d d e d
26 m i n after infection, R N A t u r n o v e r was so slow t h a t the isotope d i s t r i b u t i o n did n o t
change appreciably 25 m i n after isotope dilution.
Re/erences p. 544.
 540 L ASTRACHAN, E. VOLKIN VOL. 2 9 (1958)

TABLE I
DISTRIBUTION OF s S p AMONG R N A MONONUCLEOTIDES

Time (rain) alter: Percentage o/3sp in total R N A associated with:

~2p Dilution o[ Cytidylic Adenylic Uridflio Guanylic

addition 32p with ~zp acid acid acid acid

U n i n f e c t e d cells
3 -- 24 24 23 3°
3° 25 23 24 21 32
I n f e c t e d cells:S~P i m i n a f f e r i n f e c t i o n
3 -- 17 31 31 20
I7~ 12]/2 2I 24 27 27
3o 25 23 25 26 27
I n f e c t e d cells:S2P 2 6 m i n a f f e r i n f e c t i o n
5 -- 18 29 29 24
3° 25 2I 28 29 23

D a t a for infected cells are from e x p e r i m e n t of Fig. I. U n i n f e c t e d cells a t t i m e of i s ot ope

a d d i t i o n were a t s a m e t i t e r as i n f e c t e d cells. A l k a l i - p r o d u c e d R N A m o n o n u c l e o t i d e s w e re s e p a r a t e d
by ion-exchange procedures and their total radioactivities determined.

In another experiment designed to reduce the possibility that uninfected cells

are responsible for the initial high incorporation of 3~p into RNA, cells were first
infected in non-nutrient adsorption medium for 5 min before the addition of peptone
broth in order to allow more time for phage adsorption before resumption of meta-
bolism. Isotope was added either simultaneously with broth (at zero time) or after
intervals of 3 and 15 min. The results are shown in Fig. 2, where it is seen that when
isotope is present at zero time, the rate of ~ P incorporation into RNA was twice that
observed when isotope was added 3 min after nutrients• (A difference that m a y be
noted between the experiments of Figs. I and 2 is that, in Fig. I, isotope incorporation
essentially ceases after 3 min but in Fig. 2 it continues. Whether this difference is
13-
caused by use of different growth media (dephos-
phorylated broth or peptone broth) or by use of
preadsorbed cells for the experiments of Fig. 2 is
not known.)

>-
t-
o

O
9IK iI
-1 3-

o -
t
II

/
//
o
/
.0
/ Fig. 2. U p t a k e of s~p i n t o R N A of cells p r e v i o u s l y i n f e c t e d
I ,/
in a d s o r p t i o n m e d i u m . A t d e s i g n a t e d t i m e s a f t e r a d d i t i o n
o 3 ,6 ,5 2b
f TIME(mm)~ a t zero t i m e of p e p t o n e b r o t h t o cells p r e v i o u s l y infected
in n o n - n u t r i e n t a d s o r p t i o n m e d i u m , s s p - l a b e l e d i n o r g a n i c
p32 p32 p32 p h o s p h a t e w a s a dde d. The v a l u e s w e re a d j u s t e d as in Fig. I.
Re/erences p. 544.
voL. 29 (1958) RIBONUCLEIC
ACID TURNOVER IN E. coli 541

Experiments similar to those described in Fig. I were performed with cells grown
and infected in synthetic medium rather than dephosphorylated broth. The results
(Fig. 3) are essentially similar, in that the amounts of 3~p incorporated into RNA and
the subsequent rates of turnover are greater at earlier times of isotope addition. In
addition, the isotope distribution among the RNA mononucleotides and the change
in distribution with time is similar to those found in dephosphorylated broth. In-
creased incorporation of 32p into DNA at later times of isotope addition is again con-
comitant with decreased incorporation into RNA so that when isotope is added I i or
more minutes after infection, more s~p enters DNA than RNA shortly after isotope
addition.

~3
A
36

32

28 =
]-- ~
/I ° 36 -

~o

I TIME (m~)
,r p ~ p ~ T2 p ~ Ep)~..SS T2 p32 [XC~$S
z EXCESS

Fig. 3. T u r n o v e r of RNA-3~P a n d a c c u m u l a t i o n of DNA-3~P as a f u n c t i o n of t i m e after infection

of isotope a d d i t i o n ( s y n t h e t i c m e d i u m ) . To parallel s a m p l e s of infected cells, 82P-labeled ortho-
p h o s p h a t e w a s a d d e d a t d e s i g n a t e d t i m e s after infection. T e n m i n u t e s a f t e r 3~p addition, its specific
r a d i o a c t i v i t y w a s r e d u c e d b y a d d i n g sufficient p H 7.5 p h o s p h a t e buffer (31p) to increase t h e
o r t h o p h o s p h a t e c o n c e n t r a t i o n f r o m 22 # g of P / m l to 2250 # g of P / m l . Values for 32p in R N A a n d
D N A were a d j u s t e d as in Fig. I. T h e scale for R N A is m a g n i f i e d tenfold relative to t h e D N A scale.

Several differences are noted between experiments carried out in synthetic

medium and those in dephosphorylated broth. The ratio relating m a x i m u m counts
entering DNA to m a x i m u m counts into RNA is much higher in the synthetic medium
experiments. Furthermore, the m a x i m u m amount of 3~p incorporated into RNA is
so quickly attained in dephosphorylated broth that the rate of incorporation cannot
be measured; but in synthetic medium the rise in counts can be followed easily.
Corresponding to the slower rate of incorporation in synthetic medium is a slower
turnover after isotope dilution. A more detailed comparison of the rates of RNA
turnover in synthetic medium and dephosphorylated broth is presented in Fig. 4.
Other variations between the two media are shown in addition to the markedly
different initial rates of s2p incorporation into RNA. In synthetic medium, the
addition of excess phosphate buffer to dilute out the isotope has no immediate effect
in that subsequent incorporation of ~ P proceeds unchecked for 8 or 9 more min, and
the first evidence of 32p release is not seen until I i 92 min after isotope dilution. In
dephosphorylated broth, a decrease in 32p content is noted 4 g2 min after isotope
dilution. Not only is release of 32p delayed in synthetic medium, but once started it
proceeds somewhat more slowly and is relatively less extensive than in dephosphory-
lated broth.
In the experiments thus far described, the fate of isotope released from RNA
could not be determined because of the great excess of radioactivity always present as
Re[erences p. 544.
 542 L. ASTRACHAN, E. VOLKIN VOL. 29 (I958)

acid-soluble material. Infected cells were therefore allowed to incorporate 32p, washed
to reduce the pool of acid-soluble radioactivity, and then further incubated in nutrient
medium to follow the changes in asp content of the various chemical fractions. Upon
incubation, there were practically no changes in a2p content of the acid-soluble
material, lipids, and proteins, but large amounts of isotope left R N A and entered
DNA. From Fig. 5, which shows the changes in isotope content of R N A and D N A
after the cells were washed, it can be seen that the loss of 3~p from R N A is almost
equal to the uptake by DNA. In this experiment, no other chemical fraction could
have participated in the over-all balance of isotope since all but the acid-soluble
material contained too little radioactivity to be of significance; and whereas the acid-
soluble radioactivity was slightly higher than the radioactivity in the nucleic acids,
its changes with time were negligible in comparison to the changes of R N A and DNA.
Apparently then, the phosphorus of R N A can be incorporated into DNA, though the
pathway remains obscure".

~'3 t
*o3-
-/\
., o 9
0: T -,o g>-
F_ ->7-
>-
v- 2-
Ip > ~-

C-

=
o
Q J
J

4o S .
I ~l.~ BROTH I--
o
,o/ 2~
D

u_
T I M E OF INCUBATION A F T E R WASH ( m i n i

" 2'0 3'o io ~o ;o ~b Fig. 5. Comparison of RNA-a2P release w i t h

z
TIME (re,n) DNA-3~P accumulation in cells washed to
r e m o v e acid-soluble ~P. Three m i n u t e s after
EXCESS
infection of cells in d e p h o s p h o r y l a t e d broth,
Fig. 4. Comparison of RNA-32P t u r n o v e r in 32p-labeled o r t h o p h o s p h a t e was added. F o u r
s y n t h e t i c medium and d e p h o s p h o r y l a t e d broth. m i n u t e s later, the cells were centrifuged,
Cells were g r o w n to the same titer in dephos- washed, and reincubated to follow the isotopic
p h o r y l a t e d b r o t h and synthetic medium. Three content of R N A and DNA. Values were adjus-
m i n u t e s after infection of b o t h cultures, 3~p_ ted as in Fig. I.
labeled o r t h o p h o s p h a t e was added. Five and
one-half m i n u t e s later the specific radioactivity of o r t h o p h o s p h a t e was reduced by adding suffi-
cient p H 7-5 p h o s p h a t e buffer to increase the o r t h o p h o s p h a t e concentration in synthetic m e d i u m
from 27/zg of P / m l to 33oo #g of P / m l and in b r o t h from 6/zg of P / m l to 19oo/~g of P/ml. Values
were adjusted as in Fig. I. Different ordinates are used for the t w o cultures.

DISCUSSION

The data presented (Fig. 5) demonstrate that phosphorus released from RNA can be
incorporated into DNA. Two deductions may be made from this finding; namely, that
* I t is conceivable t h a t 3~p entering D N A came exclusively f r o m acid-soluble ~2p, with the
p h o s p h o r u s liberated from R N A serving as replacement for acid-soluble s~P. This is unlikely since
in several similar experiments, where cells were washed differently to leave greatly varying
a m o u n t s of acid-soluble radioactivity, the a m o u n t of ~2p entering D N A was always close to the
a m o u n t liberated f r o m R N A and showed no relation to the level of acid-soluble radioactivity.
Re/erences p. 544.
VOL. 2 9 (1958) RIBONUCLEIC ACID TURNOVER IN E. coli 543

the breakdown of RNA does not proceed any further than the level of nucleotides and
that ribonucleotides (or polymerized ribonucleotides) can be converted to deoxyribo-
nucleotides (or polymerized deoxyribonucleotides). If radioactive RNA were broken
down to nucleosides, then the RNA 32p would not be so nearly quantitatively in-
corporated into DNA in the presence of excess unlabeled orthophosphate.The transfer
of labeled phosphorus apart from the rest of the ribonucleotides seems unlikely since
the phosphorus involved could only be in the form of phosphate stably bound to ribose.
Preliminary results with 14C-labeled RNA also indicate the intact transfer of ribo-
nucleotide material to deoxyribonucleotides.
The question m a y then be considered whether RNA is a mandatory precursor to
DNA. From the studies of RNA turnover at different times after infection, a direct
precursor role for RNA is questionable, for, if RNA had such a role, its rate of synthesis
and turnover might be expected to increase with greater rates of net DNA synthesis,
whereas, in fact, the reverse was observed. A description of metabolic events consistent
with the data would be that RNA turnover results in the formation of acid-soluble
nucleotides incorporated into DNA, but most of the DNA may be synthesized over
pathways that do not necessarily pass through RNA.
Consideration of the metabolic events that could lead to the observed changes in
RNA turnover is pertinent. One possibility is that the rate of RNA turnover remains
constant after infection but that the RNA precursor pool becomes larger. If this were
so, then the specific activity of precursor material would fall, resulting in fewer counts
entering RNA. However, material that could reasonably be a precursor to RNA (i.e.,
the acid-soluble nucleotides) does not increase in amount for the first 30 rain after
infection and, in addition, it shortly attains the same high specific activity whether
isotope is added i or 26 min after infection (unpublished experiments). An alternate
possibility, which corresponds more closely to the data, is that very shortly after
infection a limited amount of a new kind of RNA is synthesized, and the turnover
rate of this RNA decreases with time after infection. Under these circumstances, if
isotope were added shortly after infection, most of the RNA then being synthesized
would come from labeled precursors, whereas if isotope were added at a later time,
this new RNA would have been synthesized from unlabeled precursors. This RNA
synthesized shortly after infection is considered to differ from normal bacterial RNA
because of the heterogeneous specific activities associated with the mononucleotides.
I t must be noted that the metabolism of RNA b y infected bacteria is not necessarily
fully described by considering only this new RNA synthesized shortly after infection.
In previous work where RNA was isolated from subcellular fractions ~2, it was indi-
cated that there might be two or more metabolically active species of R N A - - o n e ,
similar to that discussed here, that exhibits rapid turnover, and another that slowly
incorporates and retains isotope. This second species might account in part for the
retention of considerable isotope by RNA after isotope dilution.
In considering what role an RNA having a high turnover rate shortly after
infection might have, we recall that the work of BURTONla and TOMIZAWA AND
SUNAKAWA14 indicates that some protein synthesis must precede phage-DNA syn-
thesis and that this protein synthesis normally occurs shortly after infection. The new
RNA m a y be directly related to the synthesis of the new protein. But since incorpora-
tion of 32p into lipids and proteins is also greatest shortly after infection, the activity

Re]erences p. 544.
544 L. ASTRACHAN, E. VOLKIN VOL. 29 (1958)

of R N A at t h a t t i m e m a y n o t be d i r e c t l y r e l a t e d to synthesis of a new p r o t e i n b u t
m a y reflect a general reorganisation of cellular material.

SUMMARY

t. The t u r n o v e r of p h o s p h o r u s by R N A and p h o s p h o r u s accumulation by D N A were measured

with s2p at v a r y i n g t i m e s after infection of E. coli with bacteriophage T2. The t u r n o v e r rate of
R N A is greatest s h o r t l y after infection and decreases at later times. The reverse time dependency
is found for p h o s p h o r u s a c c u m u l a t i o n b y DNA.
2. Comparison of R N A t u r n o v e r in b r o t h and in synthetic m e d i u m reveals t h a t the R N A
of infected cells in b r o t h is m u c h more quickly responsive to changes in specific radioactivity of
o r t h o p h o s p h a t e in the medium.
3. W h e n isotope is presented to uninfected cells, the R N A mononucleotides always a t t a i n
uniform specific radioactivity, w h e r e a s within i to 4 rain after phage infection, the R N A mono-
nucleotides of infected cells have n o n u n i f o r m specific activities in t h a t adenylic and uridylic
acids have a b o u t twice the activities of guanylic and cytidylic acids.
4- When, after a period of 32p incorporation, infected cells are washed to reduce the acid-
soluble szp pool, f u r t h e r incubation results in a nearly q u a n t i t a t i v e t r a n s f e r of radioactivity f r o m
R N A to DNA.

REFERENCES

1 s. s. COHEN, J. Biol. Chem., 174 (1948) 28i.

2 E. VOLKIN AND L. ASTRACHAN, Virology, 2 (1956) 149.
3 E. VOLKIN AND L. ASTRACHAN, in \V. D. MCELROY AND H. B. GLASS, The Chemical Basis of
Heredity, J o h n s H o p k i n s Press, Baltimore, 1957, P- 686.
4 G. R. WYATT, Cold Spring Harbor Symposia Quant. Biol., 18 (1953) 133.
5 A. D. HERSHEY AND M. CHASE, J. Gen. Physiol., 36 (1952) 39.
s L. A. MANSON, J. Bacteriol., 69 (1955) lO4.
7 R. M. HERRIOT AND J. L. BARLOW, J. Gen. Physiol., 36 (1952) 17.
s M. H. ADAMS, in J. H. COMROE, JR., Methods in Medical Research, Vol. II, Year Book Publishers,
Chicago, 195 o, p. I.
9 E. P. TYNER, C. HEIDELBERGER AND G. A. LEPAGE, Cancer Research, 13 (I953)186.
10 L. ERNSTER, R. ZETTERSTROM AND O. LINDBERG, Acta Chem. Scan&, 4 (195 °) 942.
11 B. L. GRISWOLD, F. L. HUMOLLER AND A. R. MCINTYRE, Anal. Chem., 23 (1951) 192,
12 E. WOLKIN AND L. ASTRACHAN, Virology, 2 (1956) 433-
is K. BURTON, Biochem. J., 61 (1955) 473.
14 j . TOMIZAWA AND S. SUNAKAWA, J. Gen. Physiol., 39 (1956) 553-

Received February 3 r d , 1958