Kinetics of [8-14C]adenine incorporation into growing HeLa cells

510

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 8293

K I N E T I C S O F [8-14C l '~DENINE I N C O R P O R A T I O N I N T O GRC ~,r2NG H E L A C E L L S UDAI N. SINGH, RAY KOPPELMAN AND E. A. EVANS, JR.

Department o/ Biochemistry, University o] Chicago, Chicago, Ill. (U.S.A.) (Received February I2th, 1963)

SUMMARY

The time course of incorporation of [8-14C]adenine into purine bases of different RNA fractions in growing HeLa $3 cells has been studied. A high-molecular-weight RNA fraction, which resembles closely the microsomal RNA in HeLa cells, appears to be the end product in the biosynthesis of RNA. The data strongly suggest the nuclear origin of microsomal RNA in growing HeLa cells, with an RN'A fraction (nuclear RNA) present in the nucleus as a polynucleotide precursor. A simple kinetic model is proposed, which under certain restrictive conditions may be used to define the "intracellular transport rate" of RNA from nucleus to cytoplasm.

INTRODUCTION

The relationship between n- and c-RNA has been the subiect of several reports in recent years. Autoradiographic studies have provided evidence in support of the nuclear origin of the c-RNA 1-6. The interpretations of such experiments on the basis of a continuous flux of macromolecular RNA from nucleus to cytoplasm have been questioned by WATTS AND I-IARRIS7. These workers recently studied the kinetics of [8-z4C]adenine incorporation into the purine bases of n- and c-RNA in exponentially growing HeLa $3 cellss. They concluded that most of the c-RNA was synthesizc,t directly from acid-soluble precursors in the cytoplasm, and that the n-RNA was undergoing a rapid synthesis and degradation. Calculations made on the same basis as those presented here 9, indicate that the data reported by HARRIS AND WATTS 8 w e r e compatible with a precursor-product relationship between the n- and c-RNA, and that the interpretations put forth by these authors were highly questionable. In the present paper the results of kinetic studies on the incorporation of [8-z4C]adenine into various types of cellular RNA are presented. The data provide further evidence in support of a precursor-product relationship between the n- and c-RNA. MATERIALS AND METHODS

Cells The $3 clonal strain of HeLa cells adapted to horse serum was obtained from Dr. H. EAGLE of the National Institutes of Health. Stock cultures were maintained Abbreviations: c-RNA, cytoplasmic RNA; d-RNA, RNA fraction with base composition complementary to that of DNA; h~RNA, high-molecular-weight RNA; 1-RNA, low-molecularweight RNA; n-RNA, nuclear RNA.

Biochim. Biophys. Acta, 72 (1963) ,516-528

KINETIC STUDIES WITH GROWING H E L A CELLS

517

as monolayers in the growth medium (minimum essential medium) containing io ~/o horse serum described b y EAGLE1°. The spinner method of MCLIMANSet al. 11 was used for growing cells in suspension. The growth medium for suspension culture was minimum essential medium containing 5 ~ whole horse serum, modified by omitting calcium and increasing the phosphate concentration Io-fold. The cell density was kept between 2. lO5 and 7" lO5 cells/ml by daily addition of the growth medium. Under these conditions the cultures could be kept in the logarithmic phase of growth with the generation time varying from 20-30 h.

Fractionatio

oi RNA

It was observed by GIBATANI et al. TM that KIRBY'S Is phenol method for the extraction of RNA separated two types of RNA: one released into the aqueous layer (c-RNA) and the other nonextractable (n-RNA). The n-RNA could be recovered from the solid residues containing denatured proteins. Considerable evidence in support of the nuclear origin of the nonextractable fraction (n-RNA) is availableS,14-18. In the present work the RNA fractionation procedure described b y GEORGIEV et al. ~4 was used with minor changes. 0.4-0.6 ml of packed cells were extracted three times with equal volumes (5-6 ml) of water-saturated phenol (pH 6.o)-o.14 M NaC1 mixture. Phenol was removed from the combined aqueous layers by 4-6 extractions with ether. After removing ether with a stream of N~, solid NaC1 was dissolved to a final concentration of I M. The solution was kept at o ° overnight to precipitate a high-molecular-weight RNA fraction designated as h-RNA (see ref. 17). The gelatinous precipitate was washed three times with I M NaC1 solution at o °. A low-molecularweight fraction (1-RNA) was obtained from the supernatant by precipitation with 80 % ethanol-containing potassium acetate at a final concentration of 2 %. The precipitate was dissolved in water and reprecipitated twice with ethanol at o °.

Isolation o/ purine bases

The h- and 1-RNA fractions were hydrolyzed in I N HC1 at IOO° for I h and the purine bases and pyrimidine nucleotides were separated b y paper chromatography using the solvent system methanol-ethanol-conc.HCl-water (5o:25:6:19, v/v) described by KIRBYis. Purine base and pyrimidine nucleotide spots were located with. a Mineralite and cut out. Adenine and guanine were eluted with o. I N HC1 and their molar concentrations were determined from the absorbancy values at 250 and 260 m#, respectively, using the values for molar extinction coefficients given by DOROUGH AND SEATON 19.

The solid residue from the phenol extraction containing n-RBfA, DNA and proteins was washed once with 40 % ethanol and then with absolute ethanol. The n-RNA was recovered as ribonucleotides after alkaline hydrolysis (4 ml of I N KOH for 20 h at 37°). The nucleotides were purified b y adsorption from I N HC1 on activated charcoal (Norit A) and elution with ammoniacal ethanol (2 ml ammonium hydroxide-5o ml water-5o ml 95 % ethanol). The eluate was evaporated to dryness at room temperature. The purine bases were isolated as described for h- and 1-RNA. Biochim. Biophys. ,4cta, 72 (1963) 516-528

518

U.N.

SINGH, R. KOPPELMAN, E. A. EVANS JR.

Measurement o/ radioactivity o.5-ml aliquots spread uniformly over 22-mm gla~s cover slips were counted in a gas-flow-type counter. No correction for self-absorption was necessary. RESULTS

The differential labeling of the various intracellular compartments is an essential prerequisite for the calculation of transport rates from the kinetic data on the incorporation of radioactive precursors. These studies were limited to the early phase of incorporation extending over a period from 15 min after the addition of labeled adenine to 5 h, which provides the optimum condition for differential labeling of different RNA fractions. The results of one type of experiment are shown in Figs. I and 2. In order to avoid any deleterious effect on cells, no attempt was made to wash the cells prior to the addition of adenine as "chaser". In spite of some difference in the experimental techniques, the results obtained in these studies are qualitatively similar to those reported by HARRIS AND WATTS8. In contrast to a decrease in the specific activity of adenine in the n-RNA, the specific activity of guanine shows an increase (Fig. i). In h- and 1-RNA specific activities of both adenine and guanine increase with time (Figs. I and 2). Figs. 2, 3 and 4 show the results of another type of experiment in which the incorporation of [8-14C]adenine into the purine bases of 1-, h- and n-RNA was followed over a period of 2 h after the addition of [8-1~C~adenine. Rapid incorporation of radioactivity into adenine and guanine of n-RNA with no detectable lag is noticed. In contrast, the rate of incorporation of radioactivity into the purine bases of h-RNA increases with time and a "kinetic" delay is suggested by the parabolic nature of the curves for specific activities of adenine and guanine vs. time. Mathematical analysis of these curves on the basis of a simple model system strongly suggests a precursorproduct relationship between n- and h-RNA fractions in growing HeLa cells.

o

50

~

40

I

I

I

I

I

I

I

c

~ 3o A

o

o

0

30

60 90 120 Time (rain)

150

180

210

Fig. I. " C h a s e r " e x p e r i m e n t . I n t h i s e x p e r i m e n t [8-14C]adenine (specific a c t i v i t y , 0.85 mC//~mole) w a s a d d e d to cell s u s p e n s i o n a t a final c o n c e n t r a t i o n of 0.0265/~C/ml a n d t h e i n c o r p o r a t i o n w a s allowed to proceed for 4 ° m i n . A t t h e e n d of t h e labeling period n o n - l a b e l e d a d e n i n e w a s a d d e d a t zero t i m e in a m o u n t sufficient to achieve a 38-fold d i l u t i o n of labeled a d e n i n e initially p r e s e n t in t h e m e d i u m . S a m p l e s were r e m o v e d a t a p p r o p r i a t e t i m e i n t e r v a l s a n d specific a c t i v i t i e s of a d e n i n e a n d g u a n i n e in t h e t h r e e R N A f r a c t i o n s were d e t e r m i n e d . ( ~11 d e n s i t y w a s e s t i m a t e d to be 2.6. io e cells/ml. R e s u l t s are p l o t t e d a b o v e as specific a c t i v i t y ~ a d e n i n e a n d g u a n i n e in n- a n d h - R N A vs. t i m e . O - O , a d e n i n e ( n - R N A ) ; O - O , g u a n i n e ( n - R N A ) ; /x-ZX, a d e n i n e (h-RNA); [-]-[], guanine (h-RNA). Biochim. Biophys. Acta, 72 (1963) 516-528

KINETIC STUDIES WITH GROWING H E L A CELLS

519

Time (mln)(choser e×pt) 60 °

30

60

90

120

150

O50 x

~ 40 :1,

-

c

. ~ 30

-

L[

-

~ 20

0

20

40 60 Time(rain)

80

I00

Fig. 2. T i m e course of i n c o r p o r a t i o n of r a d i o a c t i v i t y into I - R N A f r a c t i o n s o b t a i n e d in t h e t w o t y p e s of e x p e r i m e n t s (Fig. z a n d Fig. 3). O - O , adenine; O - O , g u a n i n e ; e x p e r i m e n t a l details a r e i n c l u d e d in Fig. 3. LX- A , a d e n i n e ; f-q-[], g u a n i n e ; " c h a s e r " e x p e r i m e n t , details g i v e n in Fig. I.

DISCUSSION

The experimental results reported here were obtained with cells in suspension culture growing exponentially under well defined physiological conditions. GRAHAM AND SIMINOVlTCHz° have shown that in such a case cells labeled in their nucleic acids conserve the label for several generations. If it is assumed that the RNA of such cells is conserved at the molecular level and that the ceils have a constant composition, it is possible to treat the problem of the kinetics of incorporation of labeled precursors into nucleic acids in a rigorous quantitative manner. GEORGIEV and collaborators z~ demonstrated a high rate of [82P]orthophosphate incorporation into the n-R1VA of various types of cells. If conservation of RNA at a ,~ o

17o

×

150 E

18

~ 150 E ~

t4 .~-

g~7 go g_o

IiO

I0 9 .

u

<

o

90


z

70 @ 50 < 0

o "~ g 20

40

60

80

I00

Time(rain)

Fig. 3. I n c o r p o r a t i o n of [8-z4C]adenine i n t o a d e n i n e a n d g u a n i n e of n- a n d h - R N A . L a b e l e d a d e n i n e (specific a c t i v i t y , 8.45 m C ] m m o l e ) w a s a d d e d a t a final c o n c e n t r a t i o n of o . 0 2 7 / , C / m l . Cell d e n s i t y w a s e s t i m a t e d t o be 4 - 5 ' IO° cells/ml. Z e r o - t i m e s a m p l e w a s r e m o v e d 15 m i n a f t e r t h e a d d i t i o n of [8-z4C]adenine. Cells were w a s h e d twice w i t h o.14 M NaC1 a n d k e p t frozen u n t i l a n a l y s i s . O - O , a d e n i n e ( n - R N A ) ; Q - O , g u a n i n e ( n - R N A ) ; ZX-A, a d e n i n e ( h - R N A ) ; F I - [ , g u a n i n e ( h - R N A ) . T h e c u r v e s t h r o u g h t h e e x p e r i m e n t a l p o i n t s for a d e n i n e a n d g u a n i n e in n - R N A a r e c a l c u l a t e d f r o m E q n . 3 for m = o.o13 m i n - Z ( a d e n i n e ) ; A~ = 58"IO a counts]min//~mole; tSa(o) = I22. to s counts]min//~mole; ct = o . o i 9 m i n - Z ( g u a n i n e ) ; A ! = i I. io* c o u n t s / m i n / / t m o l e ; gSn(O) = 5.0" IO I counts/min//zmole.

Biochim. Biophys. Acla, 72 (1963) 5 1 6 - 5 2 8

520

U.N.

SINGH, R. KOPPELMAN, E. A. EVANS JR. 50

I

I

I

i

j

4o 6";"

-6~ 30

.

EE _m:~

u u

,o

0

20

40 Time

60

80

(rain)

Fig. 4. This e x p e r i m e n t was similar to t h a t described in Fig. 3, b u t differed in cell d e n s i t y (o.8-1.o. IO* cells/ml) and [8-1*C]adenine c o n c e n t r a t i o n (0.049 gC/ml). The c u r v e s t h r o u g h t h e e x p e r i m e n t a l p o i n t s for adenine and guanine in n - R N A are calculated f r o m Eqn. 3 for ~ = o.oi m i n - I ( a d e n i n e ) ; A~ = 53oo" lO 8 counts/min]/zmole; *Sn(o) = 17oo . Io 8 c o u n t s / m i n / # m o l e ; ¢¢ = o.oi 2 rain -1 (guanine); A g = 8oo. IOs c o u n t s / m i n / # m o l e ; gSn (o) = 2oo. lO 8 c o u n t s / m i n / # m o l e ; 0 - 0 , adenine (n-RNA); 0 - 0 , guanine (n-RNA); A - A , adenine (h-RNA); []-V1, g u a n i n e (h-RNA).

molecular level is assumed, it follows that n-RNA, with its high rate of turnover, may be considered as a precursor to some other cellular RNA fraction. The variations of specific activities of adenine and guanine with time, as seen in Figs. 2, 3 and 4 strongly suggested the possibility of a precursor-product relationship between nand h-RNA. Assuming such a relationship in the simple model system, the differential equation for the rate of change of specific activity of either base (adenine or guanine) in h-RNA is given by: dSh dt

Pnh I dH H Sn -- ~ - l - ~ Sh

(i)

where Sh is the specific activity of the base under consideration in h-RNA; Sn, the specific activity of the same base in n-RNA; H, its amount in h-RNA, and pma the amount of base transferred from n- to h-RNA per min in a certain fixed volume of culture, and

Pnh H

-

I dH H dt

The studies reported here were restricted to the early phase of incorporation of [8-14Cladenine extending over a period of 4-5 h. During this period Sn >> Sh and the second term on the right-hand side of Eqn. I can be neglected. In the following situations, often encountered in these studies, where Sn can be represented by some simple function, Eqn. I under the restrictive conditions imposed on the system is readily integrated: (i) In the "chaser" experiments, in which the initial labeling of cells by [8-14C]aBiochirn. Biophys. Acta, 72 (1963) 516-528

KINETIC STUDIES WITH GROWING H E L A CELLS

521

denine is followed by addition of non-labeled adenine, the variation of specific activity of adenine in n-RNA (aSh) can be represented by: (o) e - ~

~Sn = nSo

The integrated equation for the specific activity of adenine in h-RNA (aSh) is then given by: (2) sSh = sSla(°) + ~p~h aH ~Sn(o) 0t (x--e-~) The superscript "a" is used here for adenine. (ii) In the general situation in which the change in specific activity of adenine or guanine is given by: s, = S,(o) + A(i--e-~) (3) the integrated equation for Sh can be written as: Sh = Sh(o) + °*h ~- E(A + S. (o))t -- A( x --e-•) 1

(4)

From the knowledge of the kinetic behaviorof adenine and guanine in n-RNA, one can obtain some interesting information about the kinetics of these bases in the immediate precursor-pool of n-RNA. The differential equation for the rate of change of specific activity of any particular base in n-RNA can be written as: dSn dt

Ppa (@.~ I dN) N Sp-- - - + ~ Sa

(5)

where Sp is the specific activity of the same base in the immediate precursor-pool of n-RNA, and Ppn the amount of the base transferred from the precursor-pool to n-RNA per min. In a steady-state system, -ff =

+~-d7

=-~

~+

Since the specific activity of adenine (or guanine) in n-RNA in these studies can be represented by: sn = sn (o) + A ( i - - e - a )

the equation for the specific activity of the immediate precursor of n-RNA is given by: Sp = S n ( o ) + A

+

~--

I Ae - ~

(6)

Using the expressions derived above, it is possible to define kinetic parameters which describe quantitatively the behavior of growing cells. In Fig. 5 specific activity of adenine in h-RNA is plotted against (I-e ~t) in accordance with Eqn. 2 for ot = 2.5" lO -8 min -1 estimated from the curve of specific activity of adenine in n-RNA vs. time (Fig. I). From the slope of the straight line ap~a/aH was found to be 2.8. lO-3 min-1. Since the increase in specific activity of guanine in n-RNA can be represented by: ~S~ = gS. (o) + A ~ ( i - - e - ~ )

Biochim. Biophys. Acta, 72 (1963) 516-528

522

U.N.

S I N G H , R. K O P P E L M A N ,

E. A. E V A N S J R .

~Aw+g,%(O))t-Ag/~(l-e-dt~x10-es(guonine) 250

2 I

4

I

I

6

I

I

I

I

8 I

0

20

~b ~'~

15

c-~lO o E g~ ~' 5

~o

I

0

0.I 0.2 (l-e-=t) (odenine)

0.3

0.4

big. 5' Results in Fig. I are replotted above in accordance with Eqns. 2 and 4. The constants u s e d i n t h e c a l c u l a t i o n a r e d e r i v e d f r o m t h e s p e c i f i c a c t i v i t y o f a d e n i n e a n d g u a n i n e i n n - R N A vs. t i m e c u r v e s s h o w n i n F i g . i . ~ = 2. 5 . lO -3 m i n - X ( a d e n i n e ) ; ct = 4 . o . lO -3 m i n - l ( g u a n i n e ) ; .4 ! = 48.1o 3 counts/min//~mole; O-C), adenine; O-O, guanine.

the value for plot of SSn

gptm/SH can

be estimated graphically from the slope of the straight-line

As vs. [(Ag+SSn(o))t--~(I--e

-at

)], in accordance with Eqn. 4. The value

for sp~/SH when *¢----4.0" lO-3 rain -1, and A s = 48" lO3 counts/min/Fmole, was estimated to be 2.8. IO-s min -1 from the slope of the straight line in Fig. 5- It may be pointed out that for any arbitrarily defined value for A s ( > 4 °. lO3) one can find a value of ~ representing the experimental curve within the limits of experimental error. For example the three pairs of values: ~¢~ 4.0" lO-3 rain -1, As = 48" lO3 counts/min per /,mole; ~¢= 2.76. lO -3 min -1, Ag ----68. lO3 counts/min/#mole and c¢ ---- 2.0. lO-3 min -1, A s -----88.lO -3 counts/min/#mole are equally suitable to define the experimental curve gSn vs. time. As seen in Table I, variations in the calculated values for lag + gSn(o))t

--As( I - e -=~] are-within the limits of experimental error in such

experiments. Thus, the arbitrary choice of these parameters does not affect the value for gp~/gH to any significant extent. In the case of adenine, due to lack of a sufficient number of points, there is some uncertainty involved in the value of 0~ and hence in the value of ap~/aH which is estimated to be of the order of (2.8±0.3)" lO-3 min -1. A close agreement between the TABLE VARIATIONS

IN

gpnh/gn D U E

TO

I

ARBITRARY

CHOICE

OF

Ag

AND

I : A s = 4 8 . lO 3 c o u n t s / m i n / / z m o l e , c( = 4 . o . I o -3 r a i n -1. I I :As = 6 s - lO3 c o u n t s ] m i u / / , m o l e , c¢ = 2 . 7 6 . lO -3 m i n -x. I I I : A s = 8 8 . 1 o 3 c o u n t s / m i n / / ~ m o l e , ~¢ = 2 . 0 - l O -3 m i n -x.

Time (rain) 3o 9o 15 ° 21o

*

A

E(Ag +gSn(°))t-- ~ (z-e-ff2)] X~r°-' I o.44 1.78 3.6o 5.80

II

III

o.5o 1.8o 3.7o 6.00

o.44 1.75 3.6o 5.9 °

Biochim. Biophys. Acta,

72 (1963) 5 1 6 - 5 2 8

KINETIC STUDIES WITH GROWING H E L A CELLS

523

values for a~/aH and gl~/gH, which may be regarded as "fractional turnover rates" of n-RNA with respect to adenine and guanine, respectively, is consistent with the view that n-RNA is a polynucleotide precursor of h-RNA. Specific activities of both adenine and guanine in 1-RNA increase with time and are higher than those in h-RNA. This high rate of incorporation of radioactive adenine and guanine in 1-RNA is very noticeable in the early period of incorporation of [8-14C]adenine,and is highly suggestive of a rapid turnover of this fraction. This view is further supported by a much wider variation in the ratio of specific activity of adenine to that of guanine as compared to such variation in h-RNA. The high incorporation rate can be attributed partly to the cellular transfer RNA which is known to undergo rapid exchange of end-group nucleotides with the intracellular pool. However, the possibility of a metabolically active RNA fraction incorporating labeled adenine and guanine into intrapolynucleotide positions cannot be excluded. The heterogeneity of this RNA fraction and the nature of the process involved in the incorporation of labeled precursors are being investigated currently. In the second type of experiments (Figs. 3 and 4) since specific activities of adenine and guanine in n-RNA are much higher than those in h-RNA, the simplified expressions derived above are also applicable. As seen from Figs. 3 and4 the variation of specific activity of adenine (aSh) or of guanine (gSn) in n-RNA can be represented by Eqn. 3. Assuming n-RNA as the precursor of h-RNA, specific activities of adenine (~Sh) and guanine (gSh) in h-RNA are given by Eqn. 4. The values of aprm/*'Hand gpnb/gH a r e estimated to be I.O. lO-3 min -1 and 0.8. lO-3 min -1, respectively, from the slopes of the straight lines in Fig. 6 in accordance with Eqn. 4. Fig. 7 shows a similar plot of the results obtained in another experiment under identical culture conditions, except for different cell density and [8-14C]adenine concentration in the medium

0

EAg + gsn (O))t-Ag/: (l-e-:t)7 x 10 -5 (guanine) 4 8 12

I

,

r

,

I

16

I/[ 16 ~"

16

× ID

b x

_e

t2

12 ~ C

E E

o < Z

.12

4

~

v

o <

i

I

4

t

I

8

t

I

IZ

i

-(Ao+°Sn(O))t-Ae/,(I-e-~t'~ x 10. 6 (Qclenine)

o

16

Fig. 6. Results presented in Fig. 3 are replotted in accordance with Eqn. 4. The constants used in the calculation were derived from the curves for n-RNA shown in Fig. 3. © - O , adenine; 0 - 0 , guanine.

Biochim. Biophys. Acta, 72 (1963) 516-528

524

U. N. SINGH, R. KOPPELMAN, E. A. EVANS JR.

~Aa+"Sn(O))t-A"/<[(I-e-atlx lO-7(°denine) x

"6 40 I E

5

I0

15

20

I

I

,

,

25

y

30

5540 ®

,

7

~3o ~ 2o

20 ~ o

~ ,o o, o 0

I

I

I

I

I

I

,o

20

~o

4o

~o

~o

~ ~o

~,+ o~.~o>,,-,,/<.o-~-"71.,o-°c,<=.~.~> i

Fig. 7. Results presented in Fig. 4 are replotted in accordance with Eqn. 4. 0 - 0 , 0 - 0 , guanine.

adenine;

(Fig. 4). The values of pnh/H for adenine and guanine estimated as 0. 9- lO -3 rain -1 and 0. 7. lO -8 min -J, respectively, are comparable to those obtained in the earlier experiment (Figs. 3 and 6). A close agreement between the values for pnh/H for adenine and guanine in both the experiments leaves little doubt that the high "turnover" rate of n-RNA is primarily due to this RI~A fraction acting as a polynucleotide precursor to h-RNA, and not to its rapid breakdown as suggested by HARRIS AND WATTS s.

Under the restrictive conditions imposed on the theoretical model system, I dH p ~ / H -- H dt -- k, where k is the rate constant for the exponentially growing cultures. Since the adenine/guanine ratio in h-RNA is constant, the "fractional turnover rates" of n-RNA with respect to adenine and guanine should be the same. The generation time of cells in these studies varied from 20-30 h and corresponding values for k varied from (o.38-o.58).1o -3 min -1. The observed values for apnh/aH (0.9 and I.O" lO -3 min -1) andgpnh/~H (0. 7 and 0.8, lO -3 rain -1) are too high as compared to the values of k estimated from generation time of cells. This discrepancy m a y be due to either (i) the unstability of h-RNA or (ii) the existence of some other precursor to h-RNA besides n-RNA. The existence of an enzyme or enzymes involved in the transfer of polynucleotides from the soluble to microsomal RNA has been demonstrated by several workers 2~-*s. Metabolically active soluble RNA has been shown to be the major constituent of the 1-RNA fraction ~7. It is further noticed that during the early period of incorporation the ratio of the specific activity of adenine to that of guanine in the I-RNA is much higher than in h-RNA. Thus, the transfer of polynucleotides from 1-RNA to h-RNA will also explain the difference in the estimated turnover rates of n-RNA with respect to adenine and guanine. As pointed out earlier pnh/[I m a y he regarded as the "fractional turnover" rate of n-RNA. The values of pnh/H for adenine [(2.8±0.3)" IO-3] and guanine (2.8. lO -3 rain -1) estimated from the "chaser" experiment are unusually high as compared to the corresponding values obtained in the second type of experiments. The reason for this large discrepancy is not clear. Since adenine at concentration higher than lO -3 M has been observed to have a toxic effect on cells, it is probable that addition of non-labeled adenine in these experiments interferes with the normal nucleic acid B%chim. Biophys. Acta, 72 (1963) 516-528

KINETIC STUDIES WITH GROWING H E L A CELLS

525

metabolism. The quantitative significance of this constant, and its relationship with the nucleic acid metabolism under varying physiological conditions are being investigated. Assuming the conservation of n-RNA at the molecular level as suggested from a close agreement in the values of praa/Hfor adenine and guanine, the behavior of its immediate precursor during the experimental period is given by Eqn. 6. The most likely immediate precursors of n-RNA are probably the triphosphonucleotides in the nucleus. Theoretically, the validity of Eqn. 6 can be tested by determining the specific activity of adenine (and guanine) in ATP (and GTP) obtained from the nucleus. In practice, however, such an attempt is confronted with several technical difficulties, one of these being the limited amount of sample available. It is a reasonable assumption, that various low-molecular-weight derivatives of adenine (nucleosides and nucleotides) are in rapid equilibrium with each other, and the average specific activity of adenine in such a pool may be regarded as an estimate of its value in triphosphonucleotides. In one experiment the supernatant obtained after the precipitation of 1-RNA with 80 ~/o ethanol was evaporated to dryness. The powder was dissolved in I.O N HC1 and treated with "Norit A". The adsorbed material on the charcoal was eluted, evaporated to dryness and heated in I N HC1 at IOO° for I h. Paper chromatography of'the hydrolysate as described earlier revealed five spots under ultraviolet light with Re values 0.32, 0.46, o.54, 0.67 and 0.75. Fig. 8 shows a radioactivity scanning of the I

"2

i

I

Origin

I

!

3

I

I

I

I

I

0.32

0.46

0.54

0.67

0,75

Fig. 8. Radioactivity scanning of the chromatogram of the "alcohol-soluble" pool in the solvent system described in the text. Ultraviolet-absorbing spots w i t h R F values o.3e and o.46 have been identified to be due to guanine and adenine, respectively.

chromatogram for the zero-time sample (I5 min after the addition of [8-1~C]adenine). Peak i corresponds to guanine and Peak 2 to adenine. A significant amount of radioactivity (Peak 3) is associated with the spot corresponding to an Rp value of 0.67 . Adenine was eluted from paper and its specific activity was determined. Table II shows the experimental values for the specific activity of adenine, which are compared with the values calculated from Eqn. 6, for ,t = o.o124 rain -1, Aa ----12o- IOs counts per apma min/#mole, aH -- 1.6. lO-3 min -1, aSn(o ) = 31" IOs counts/min/pmole (Fig. 9). Biochim. Biophys. Acta, 72 (I963) 516-52e

526

o . N . SINGH, R. KOPPELMAN, E. A. EVANS JR. TABLE II

COMPARISON OF CALCULATED AND EXPERIMENTAL LOW-MOLECULAR-WEIGHT

VALUES

PRECURSOR

OF SPECIFIC POOL

Specific activity o! adenine in the immediate precursor pool o] n-RNA calculated from Eqn. 6 (X zo -s)

Time

(,tin)

H

o 15 45 lO5

~

271 251 218 183

IN

Speci[ic activity o] adenine in alcohol-soluble pool (counts/rain/

H = 3

ACTIVITY OF ADENINE

OF R~A

= 2

l~mole X z o - 3 )

343 311 260 203

352 382 316 175

° s°(o) +A~)*-Ao/=(I-~-~t~,I0-~ o

o

a2o

2

4

6

8

I0

i

]

I

I

I

~7 x

6 IOC E --~ 80 E

3o

2s ! z~

Iz

4o

2o

"~:

<

0

0

1 20

I I 40 60 Time (rnin)

I 80

I I00

-o

I0 <

Fig. 9. Incorporation of [8-14C]adenine into adenine of n- and h-RNA. This experiment was similar to that described in Fig. 3. Cell density 2-3' IOe cells/ml. [8-1*C]Adenine (specific activity 8.45 mC/mmole) added at a final concentration of o.oi8pC]ml 15 min before the removal of zero-time sample. O-(D, specific activity of adenine in n-RNA vs. time. The curve through these points is the theoretical curve calculated from Eqn. 3 for aSh(o) = 3I'IO s c o u n t s ] m i n / p m o l e ; Aa = 12o" IOs counts]min//zmole, and ~ = o.oi24 rain-1. Q - O , specific activity of adenine in h-RNA vs. F ( A a + aSu(o))t ----Aa (i_e_a)7 . "pnh/aH is estimated to be 1.6.io -8 min-x from the _J L slope of the straight line. I n spite of the assumptions i n v o l v e d in the d e r i v a t i o n of Eqn. 6, a n d the fact t h a t the e x p e r i m e n t a l values are o n l y estimates of the specific a c t i v i t y of adenine in the immediate precursor-pool of n - R N A , the calculated a n d e x p e r i m e n t a l l y d e t e r m i n e d values are of the same order of m a g n i t u d e . This provides a d d i t i o n a l support to the hypothesis t h a t the high t u r n o v e r rate of n - R N A is p r i m a r i l y due to its rapid conversion to h-RNA, rather t h a n due to its rapid breakdown a n d resynthesis as suggested b y HARRIS AND WATTSs. RAKE AND GRAHAM24 have recently reported on the kinetics of [14C]uridine incorporation into I- a n d h - R N A of growing Strain-L cells. T h e y concluded t h a t h - R N A was the end p r c d u c t in the biosynthesis of R N A in growing cells. This is consistent with the results reported here on the kinetic behavior of h - R N A in growing H e L a cells. The role of 1-RNA as the possible i m m e d i a t e precursor to h - R N A , as Biochim.

Biophys..dcta,

72 (1963) 516-528

KINETIC STUDIES WITH GROWING H E L A CELLS

527

suggested by RAKE AND GRAHAM ~ , is not well documented. Although transfer of some radioactivity from 1- to h-RNA, probably due to exchange reactions, is not excluded, quantitative considerations indicate that 1-RNA is probably not the major intermediate in the biosynthesis of h-RNA. The origin of ribosomal RNA has been the subject of considerable interest in recent years. MCCARTHY et al. 25-28 demonstrated the existence of two sequential precursors (eosome and neosome) in the synthesis of ribosomes in growing Escherichia coll. KITAZUME et al. z9 postulated that an RNA fraction (d-RNA) with a base composition complementary to that of DNA might be a primary polynucleotide precursor to yeast ribosomal RNA. Similarity in base composition and low turnover rate of h-RNA and microsomal RNA in I-[eLa cells strongly suggest an identity between these two RNA fractions. These studies suggest that the microsomal RNA in growing HeLa cells is derived from a polynucleotide precursor (n-RNA) in the nucleus, in accord with the conclusions of ~V~cCARTHY e t a / . 25-28 and KITAZUME et al. 29 o n the origin of ribosomal IZNA in growing E. coli and yeast, n-RNA has been shown by GEORGIEV et al. 3° to consist of two RNA fractions, a nuclear ribosomal RNA with base composition similar to that of c-RNA and an RNA with base composition complementary to that of I)NA. From the present studies it appears that the nuclear ribosomal RNA is probably the immediate precursor of microsomal RNA in HeLa cells. The nuclear ribosomal RNA may be synthesized directly from acid-soluble precursors using DNA 31 or RNA 32 as the template, or, in accordance with the hypothesis proposed by KITAZUME et al. 29, d-RNA may be the immediate precursor of nuclear ribosomal R N A . No direct experimental evidence in support of d-RNA as the "messenger" or "information" RNA is available. Such a role of d-RNA is merely inferred from its resemblance to DNA in base composition and its relative instability 3a. It is well known that in non-growing cells all types of RNA including h-RNA undergo degradation to some extent, and the "instability" of d-RNA in such a culture may be due to its breakdown. In growing cultures, this apparent "instability" may be primarily due to the conversion of d-RNA into ribosomal RNA. The accumulation of the d-RNA fraction in the so-called "step-down" culture of HAYASHI et al. 34 can then be explained on the basis of a partial block of the conversion of d-RNA into ribosomal RNA. Further work on the function of d-RNA as the possible intermediate in the synthesis of nuclear ribosomal and microsomal RNA in growing HeLa cells is in progress. ACKNOWLEDGMENT

This work was supported in part by a grant from the National Foundation. The authors are indebted to Mrs. R. TIMMONS for her invaluable technical assistance during this study. REFERENCES i 2 a 4 5 6 ~'

L. D. p. L. M. R. J.

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Biochim. Biophys. Acta, 72 (1963) 516-528