action of ribonuclease upon the polysomes of cultured mouse cells

I~r VITro Vol. 8, No. 1, 1972

ACTION OF RIBONUCLEASE UPON THE POLYSOMES OF CULTURED MOUSE CELLS ~

K. RICHARDS, P. A. KITOS, AND i~. T. HERStI

Department o] Biochemistry, University o] Kansas, Lawrence, Kansas 660/~

SUMMARY

Pancreatic ribonuclease (RNase) and 3H-uridine were used to study certain com- positional and ontogenetic features of the polysomes of strain L mouse cells. Growing cells were exposed to the radioactive nucleoside, "H-uridine, for brief defined periods, and the sensitivity of the polysomes to digestion by RNase was determined. The RNase- resistant RNA of polysomes is shown to be primarily ribosomal, and the RNase-sensitive material formed during brief pulse labeling studies is largely messenger RNA. Actinomy- cin D inhibition of RNA synthesis was used to confirm this identification.

The technique described here was used to investigate the effects of hydrocortisone on polysome formation. The hormone (10 -~ M) lessens the extent of the nucleoside incorporation into polysomal and total RNA and delays the appearance of newly synthesized 18 S and 28 S rRNA into cytoplasmic polysomes.

Pancreatic ribonuclease (RNase) at low concentrations degrades polysomes almost quantitatively to particles of the size class of monosomes (1-4). When digestion of retieuloeyte ribosomes is performed, even at high concentrations of RNase, only a relatively small fraction of the ribosomal RNA (rRNA) is released into the sur- rounding fluid as low molecular weight material, even though the rRNA has been extensively damaged (5). Apparently most of the breaks are obscured by the secondary structure of the RNA moiety and by protein-RNA interactions which preserved the over-all integrity of the ribosomal partMe. As much as 30% of the RNA may be removed from the ribosome without altering its sedimentation coefficient or its appearance in the electron microscope (5).

In the present, study the action of RNase is reexamined from the point of view of using the enzyme as a tool for measuring, quantitatively, changes in the polysome fraction of animal cells which have been cultivated in vitro. The technique was applied to two eases: (a) to the effect of aetinomycin D on RNA synthesis in order to

* This work was partially supported by grants from the United States Public Health Service (GM 10866), from the National Science Foundation (GB 13924), and from The University of Kansas Gen- eral Research Fund.

verify the assignments of RNase-sensitive RNA to the mRNA class and (b) to the effects of hydrocortisone on the synthesis of RNA contained in the polysomes.

MATERIALS AND METHODS

A subline of NCTC clone 929 mouse ceils, strain L (6), was used in all experiments reported here. The cultures were maintained in the manner described previously (2) as mono- layers on all interior surfaces of 250-ml milk dilution bottles (MDB) or 11 • 34 cm roller vessels. Experiments were generally performed on 6-day-old cultures with six MDB's (approxi- mately 5 x 108 cells) being used per experiment.

In the tracer studies carried out at 37~ uridine-53-H (specific activity 21.7 Ci per mmole; New England Nuclear Corp.) was added directly to the nutrient medium. The amount of isotope in the experiments varied somewhat, de- pending upon the length of the labeling period, but generally fell in the range of 1 to 10 t~Ci per ml of medium.

Before disrupting the cells, the nutrient medium was decanted from the culture vessel, and the monolayer was washed with 0.85% sodium chloride solution. Five milliliters of TSM buffer (0.05 ~ Tris, 0.01 ~ MgSO,, adjusted to pH 8.5

48

L CELL POLYSOMES 49

at 0~ with HCI) containing 0.5% Nonidet P40 (Shell Chemical Company) were swirled gently over the monolayer. The detergent disrupts the cell membrane but leaves some debris, including the nuclei, attached to the walls of the culture vessel. The extract, which contains the cytoplasmic polysomes, was centrifuged at low speed to remove residual cell debris. These and all subsequent steps were carried out at 0 to 2~

The polysomes were partially purified by centrifugation in a 30 rotor of the Spinco model L2 preparative centrifuge (3 X 10' rpm for 100 min). In some experiments the polysomes were sedimented through a 2-ml 2 ~ sucrose pad containing TSM buffer (3 • 10' rpm for 300 min). Both procedures yielded preparations which produced similar polysome optical density profiles in sucrose gradients. The polysomal pellet and the sides of the centrifuge tube were rinsed thoroughly with buffer, and the polysomes were dispersed with a glass rod in an appropriate volume of TSM buffer. If necessary, a hand homogenizer equipped with a loose-fitting Teflon pestle was used to disrupt any microgels. Whether the polysomes were resuspended by diffusion or by this procedure, there was no dif- ference in the quality of the polysomal profile. The solution was clarified by low speed centrifugation (40 rotor in a Spinco model L2, 10,000 rpm for 7 min). So prepared, the solution of polysomes had an absorbanee ratio (260/280 m/~) of 1.72 to 1.82 and a minimum at 236 mt~.

The polysomes contained in a 0.2-ml aliquot portion were resolved by centrifugation (Spinco SW 50.1 rotor at 50,000 rpm for 38 rain) through a 10 to 45% (w/v) linear sucrose gradient (5.4 ml) containing TSM buffer and 5 • 10 -'~ ~r KC1. The gradients were eluted with an Isco model 180 density gradient fractionator while absorbance at 258 mt~ was monitored con- tinually with an Isco model UA-2 ultraviolet analyzer.

In order to test the susceptibility of polysomes to digestion with RNase, a series of Spinco 40 rotor centrifuge tubes was prepared containing pancreatic RNase (Pentex Corp. 5• crystallized) in amounts ranging from 0 to 60 ~g per ml. One milliliter of the polysome solution was added to each tube. The tubes were immedi- ately placed in a cold 40 rotor and centrifuged at 0~ for 90 min at 36,000 rpm. In the absence of RNase this eentrifugation is more than 96%

efficient in pelleting the ribose-containing material of the extract. The supernatant fluid from each tube was poured into an 8-mm test tube, the mouth of which could be fitted tightly over the mouth of the centrifuge tube. Thus fitted together the inverted 40 rotor tube was centrifuged in the swinging bucket head of a refriger- ated International centrifuge for 2 rain at 300 RCF. The pellet and the sides of the centrifuge tube were washed with 1 ml of buffer and drained in the same manner. This procedure results in the quantitative removal of the supernatant liquor from the 40 rotor tube. Pentose determinations on samples of the supernatant fluid were made by the orcinol method (7) using D-ribose as the standard.

For radioactivity measurements the pellet was allowed to resuspend in water. The liquid was quantitatively transferred to scintillation vials and taken to dryness. The residue was dissolved in 200 td of Hyamine hydroxide (Packard Chemical Co.), and then 10 ml of a scintillation cocktail composed of 0.3% p-terphenyl and 0.005% dimethyl POPOP in toluene were added. Radioactivity measurements were made in a Packard Tri-Carb scintillation spectrometer, model 3310, and the results were corrected for quenching using an external standard.

It has been reported that polysomes from L cells broken by mechanical means are contami- nated with rapidly labeled nuclear RNA (8) This causes an elevation in the specific activity of the polysome preparation. If a nonionic detergent, Triton X100 (0.05%) is used to break the cells, contamination can be avoided. In this laboratory 0.05% Triton X100 did not give good cell breakage, and the yield of polysomes was only l0 to 15% of that obtained when 0.5% Nonidet P-40 in TSM was used to disrupt the cells. However, polysomes prepared by either of the disruptive procedures had the same specific radioactivity. Therefore, if the Triton method of Perry and Kelley (8) results in polysome preparations tlmt are free from nuclear RNA, then in the present study nuclear contamination must likewise be minimal.

RNA was isolated from polysomes essentially by a sodium dodecyl sulfate method (9). The polysome solution was pipeted into a test tube at 37~ containing 0.11 volume of the following mixture: 5% sodium dodecyl sulfate (Duponol C, DuPont Co.), 0.1% Bentonite, and 0.5 M Tris

50 RICHAItDS, KITOS, AND HERSIt

0 RNAosA1

04 0

C 30 ~ug/ml RNAose

Jk__ TOP

TOP

O 60 .ug/ml RNAase

Jk TOP

FIG. 1. The effect of pancreatic ribonuclease upon the distribution of polysomes in sucrose gradients.

(pH 7.5 adjusted at 0~ After 30 see of gentle agitation the mixture was chilled to 0~ One milliliter of it was layered onto a 28-ml 5.8 to 22.6% (w/v) sucrose gradient containing 0.1 Ivz NaC1 and 0.01 M Tris, pH 7.5. After centrifugation for 15 hr at 4~ at 25,000 rpm in an SW 25.1 rotor (Spinco) the gradient was fraction- ated as described. When necessary, 1-ml samples of the effluent were collected for radioactivity measurements. Each fraction was mixed with an equal volume of a solution containing 1 mg per ml yeast RNA and 0.5 mg per ml bovine serum albumin, and the resulting mixture was made 5% with respect to triehloroacetic acid (TCA). The precipitate was collected on glass fiber filters, washed twice with 5% TCA, twice with 95% ethanol, and dried. The filters were placed in scintillation vials and analyzed as described.

Hydrocortisone (Sigma Chemical Co.) was kept as a 5 • 10-" M solution in 95% ethanol. Actinomycin D, a gift of the Research Labora- tory of Merck, Sharp and Dohme Co., was dissolved in 70% ethanol at a concentration of 400 t~g per ml and was stored at --20~ in the dark. Bentonite was purchased from Fisher Scientific Co. The procedure of Peterman and Pavlovee (10) was used to obtain the proper size class of Bentonite for the inhibition of RNase activity.

]:~E S U L T S

Effects of ribonuclease on polysomes. In a typical preparation of polysomes from L cells (6 days after transfer and 2 days after nutrient replenishment), most (60 lo 70%) of the particles sediment faster than the ribosomal dimer

(Fig. 1A). If the preparation is treated with RNase before gradient centrifugation, the amount of material in the polysomal region diminishes, and the characteristic polysome peaks disappear (Fig. 1, B, C, D), while the magni- tudes of the monosome peak and the lower molecular weight material near the top of the gradient increase. Athough it has not been identi- fied, the material which remains in the polysomal region after RNase treatment could be ag- gregates of ribosomes damaged by the enzyme (5, 11).

Polysomes isolated from L cells which had been exposed to 3H-uridine for 30 min were subjected to RNase digestion as described (60 ~g RNase per ml, 0~ 30 min). This treatment resulted in the release in the form of acid- soluble substances of 59.0 • 2.2% (SD of mean) of the radioactivity which had been in- corporated into the polysomes, but at the same time it liberated only 14 • 2% of the total RNA (determined by pentose analysis). The fractional release of rapidly labeled oligonucleotides by this digestion was not affected by the concentration of polysomes, but the release of orcinol-positive constituents was found to increase as the concentration of polysomes decreased. Within the concentration range of polysomes used in this study (0.18 to 0.45 mg per ml) the variation in the amount of pentose released was not significant. The release of pentose-containing material was found to be directly proportional to the amount of enzyme used in the digestion, but at RNase concentrations greater than about 10 t~g per ml the liberation of rapidly labeled components

L CELL POLYSOMES 51

from the same polysomes was independent of further enzyme increase (Fig. 2). Consequently, 60 RNA components derived from the po]ysomes by high concentrations of RNase were much lower in specific radioactivity than those derived at low concentrations of the enzyme�9 4 0

The fraction of the polysomal radioactivity "' dmt is released by RNase at 60 /~g per ml de- < pends upon the duration of the labeling period .a (Fig. 3). After a 5- or 10-rain pulse, 80% of the ~:20 polysomal radioactivity was sensitive to the enzyme, and the average specific activity of the IlNase-sensitive RNA was found to be 23 times that of the RNase-resistant RNA. As the labeling period was extended, the fraction of the radioactivity lhat was released declined sharply at first and then leveled off (Fig. 3).

Darnell and his associates (12-14) demon- strated that, in HeLa cells, there is a 20- to 60-rain lag between the synthesis of the 18 S and 28 S rIINA precursors and the actual appearance of mature subunits in cytoplasmic ribosomes.

Likewise, in L cells, new 18 S rRNA is present in significant amounts after 30 min although

80 new 28 S RNA takes somewhat longer to appear (Fig. 4). By 2 hr both types of newly synthesized rRNA are present in abundance. Notably, the incorporation of RNase-resistant RNA into o cytoplasmic polysomes and the appearance of <7 newly synthesized rRNA follow a similar time cc course. Thus, the newly formed RNase-resistant m ~ RNA appears to be at least part ly ribosomal, c~ The relative insensitivity of the rRNA to RNase m w 6 0 < does not imply that it is intrinsically immune to w this digestion or even that it is not digested. In fact, rRNA of mature ribosomes is extensively hydrolyzed by RNase (Fig. 5). Noneovalent in- '" teractions are capable of retaining most of the o

cO

fragments within the ribosome structure (5). >- __l

Effects o] actinomycin D on polysomal R N A . o a_ At low concentrations, actinomycin D preferen- z 4 0 tially inhibits rRNA synthesis (15-17 and Fig. :~ 6) The concentration-dependent effect, of acti- a _

�9 O

nomycin D on the labeling of L cell cytoplasmic polysomes with "H-uridine is shown in Fig. 7. The cultures were pretreated for 20 min with 0 the antibiotic and then pulsed with "H-uridine. The fraction of the radioactive polysomal material which is RNase-sensitive rises with increas- ing concentration of actinomycin D to a maxi- mum of 80% at 0.08 /xg antibiotic per ml, the lowest concentration capable of completely in-

I t j

/ ~ ~ o o - -

/

O 0 t ~ t t t t 2 0 40 60

RNAese CONC.(pg/ml)

Fro. 2. The release of low molecular weight material from L cell polysomes by various concentrations of ribonuclease. The cells had been pulse- labeled for 30 min with '~H-uridine. O, radioactivity released; A. RNA released as measured by ribose determinations.

I I I I

r hydrocortisone teated

I 1 I I 30 60 90 120

LENGTH OF LABELING PERIODIminJ

FIG. 3. The percentage of the radioactivity in polysomes released by RNase as a function of the labeling time. @, control; O, cells exposed to hydroeortisone. See section on effects of hydrocortisone upon the pulse labeling of polysomal RNA.

~52 /tICHARDS, KITOS, AND HEIISH

i N

0

I.(:

TOP

I00

0

3000 ~"

TOP

D

TOP TOP

500

O "O

T

FIG. 4. Sucrose gradient patterns of RNA isolated from polysomes after various pulse periods. A, 10-min pulse with 10 gCi per ml in the medium ; B, 30-min pulse, 5 ~Ci per ml ; C, 60-min pulse, 5~Ci per ml ; D, 120-rain pulse, 0.2 gCi per ml (see text).

hibiting rRNA synthesis in L cells (17) (Fig. 6). As shown in Fig. 8, if the cultures were pulse-labeled for only 10 rain, the antibiotic had no effect on the release of radioactive polysomal material by RNase. Thus, the RNase-resistant polysomal RNA formed during 30- to 120-min labeling periods must be primarily rRNA, whereas the 20% RNase-resistant radioactive polysomal RNA which accumulates during short (5- to 10-min) pulses or after treatment with 0.08 ~g per ml aetinomycin D must not be rRNA. This concentration of antibiotic does not inhibit tile synthesis of tRNA or 5 S RNA, both of which should be present in the polysome preparations. Although little is known about their sensitivity to RNase, retention of part of the tRNA or 5 S RNA by the ribosomes after treatment with the enzyme could account for some or all of the residual RNase-resistant radioactive material. Alternatively, the RNase-resistant radioactivity may be associated with regions of mRNA protected from the enzyme by proximity to the ribosome (18).

Labeling o[ polysorne-associated tRNA, 5 S RNA, and 7 S RNA. To what extent does polysome-associated tRNA become radioactive during short labeling periods? Owing to limitations in the amount of material available and difficul-

ties in separating tRNA from possible mRNA fragments, an indirect approach was used to an- swer this question. Polysomes from 30-rain- pulsed cells were purified, and their specific radioactivity (cpm per ~g ribose) was determined. The low molecular weight RNA of the postmi- crosomal supernataut fluid should be primarily tRNA. Protein was removed from the fraction by phenol extraction, and the RNA was precipi- tated with 0.25 ~ HCIO,. The specific activity of this fraction was determined. Using this value and considering that, by calculation, approxi- mately 2% of the RNA of functional monosomes is tRNA, we determined that the polysome-associated tRNA could account for no more than 5% of the total radioactivity of the polysomes. Using the available data for the quantity of each species of RNA per cell (19) and several reason- able assumptions, we calculated the rates of synthesis of the various RNA's in L cells (Table 1). According to these calculations it appears as though new tRNA and mRNA appear in the cytoplasm at similar rates. However, since only about 10% of the total tRNA is polysome-associated whereas essentially all the mRNA is polysome-bound, the rate of appearance of newly synthesized tRNA in the polysome could be only 5 to 6% of the rate of appearance there of

L CELL POLYSOMES 53

mRNA. Since the rate of appearance of 5 S ]INA (19) and 7 S RNA (20) is even lower, the combined new tIINA, 5 S IINA, and 7 S RNA ~J.o should account for 10 to 15% of the polysomal radioactivity at most. With more lengthy pulse intervals, as significant amounts of radioactive rRNA have begun to appear, the fractional con- tribution of the slower sedimenting RNA species o would be even less.

The validity of the calculations from which the values of Table 1 are derived depends upon 2.c the accuracy of the estimates of the half-lives of the various RNA species and the amount of mRNA per cell. Although the actual values ~t.c should be considered with caution, the), and the experimental results indicate that the two large ribosomal IINA forms (18 S and 28 S) and

0 ~D 04

oa5

CO~ROL

' ' ' T 'OP

'O cD oJ

R N ~ TREATED B

TOP

Fro. 5. Effect of pancreatic RNase on the isolated polysomal RNA. The ribonucleie acid was isolated by the sodium dodecyl sulfate treatment and subjected to centrifugation on a 5 to 20% (w/v) sucrose gradient (0.1 M NaC1, 0.01 ~r Tris, pH 7.5). A, control; B, RNA from polysomes which had been previously subjected to treatment with 60 ~g per ml RNase at 0~ for 60 min.

000 o

500

TOP

TOP

c~ 70

I000

2.(3

OO8 #gel o0 LO o4

" L O O

c) 70

' 000

TOP

Fie. 6. Effect of acdnomycin D on pol3=,-omes pulse-labeled for 60 rain with ~H-nridine. A, con- trot; B, cells treated with 0.04 ~g per ml actinomycin D for 30 rain prior io labeling period; C, poIysomes from ceils lreaied with 0.08 ~g per ml actinomycin D for 30 rain prior to labeling period.

mI~NA comprise tile bulk of the labeled RNA of polysomes.

Effect of hydrocortisone upon the pulse labeling o] polysomal RNA. The glueocorticoid hormone, hydrocortisone, retards the growth of cultured bone cells and elicits profound changes in their RNA and protein metabolism (21, 22), In like manner it slows the proliferation of L cells (23, 24). We have observed that treatment of L cells with 10 -~ ~ hydrocortisone for 24 hr greatly diminishes the incorporation of ~H-uridine into polysomes (Table 2) but causes only minor changes in the polysome profile. Incorpo- ration of "H-uridine into total RNA is likewise diminished by 40%, but uptake of the precursor by the cells is only slightly affected (Fig. 9).

The effect of hydroeortisone treatment (10 -B

54 t / ICHAt /DS, KITOS. AND H E t l S H

M, 24 hr) upon the RNase sensi t ivi ty of labeled polysomes was tes ted (Fig. 3). I f the cul ture had been pulsed-labeled with ~H-uridine for 30 min, 74 -+- 4% of the radioact ive subs tance incorpora ted into the polysomes of hormone- t r ea t ed cells was RNase-sens i t ive versus 59 +_ 2.2% for the controls.

Al though the differential incorpora t ion of ra-

. . . . . . 2 ti\

6 o 5 o

0 ~ 4 0 I i I I I I I J J 0 0.1 0.2 ~o

ACT. D CONC.~g /m l )

FIG. 7. Tim effect of various conccntralions of actinomycin D upon the sensitivity to ribonuclease digestion of Ihe ra(tioactive material which had beeomt, associaled with poly,,omal t lNA dttring a 60-min pulse with :~H-uridine. The culha'cs were preincubated wilh aclinomyein D for 20 rain prior to athninislrat ion of the radioactivity precursor. e , % of total polysomal radioactivity released by I tNase; O, specific activity of isolated polysomcs prior to RNase t reatment (cpm per mg poly~omes • 10-'~).

dioaetive ur idine was originally in t e rp re t ed as a n effect of the ho rmone on the relat ive rates of

TAB LE 1

I{ATES (IF ~ Y N T n E S I S OF S(IMI,; C Y T ( H ' L A S M I C

IINA Sm.:cH.: s

Species

18 S q- 28 S r I /NA

5 S IINA tRNA 7 S I /NA mRNA

No. of No. of Nucleo-

Molecules tides of Each of Each Species Species per Cell (N 10 -6) per Cell

( • 10 ~0)

5.06 3.4 + 5 06"

5.6" O. 07 100" 0.80

5 06 0.07 0.17 ~

Rate of Rate of Synthesis I Appear-

cleotides per min. per min. per Cell per Cell (X 10 -s) X 10 b)

1896

3,9 I' 44 b

3 . 5 c

4 . 4 d

74 91,,

,, Ref. 19. t, Calcula ted on the assumpt imi t h a t these

species are metabolical ly s table and double in amount once during the douhl ing t ime of the cell (30 hx').

Calculated assuming one molecule [)nund per ribosome.

'~ Calculated assuming two molecules hound per ribosome.

Calcula ted on the asstllnl)tion t h a t the mass rat io of r l INA to ml lNA is 20:1 (llef. 26).

/ Calculated from the et luat ion In 2 (pool size)

ra te of synthes is = half-life

with the half-life taken as 130 to 160 min (Ref. 2). ~ Calcula ted assuming t ha t all ml INA is fnui~d

in p()lysomes (fief. 12).

01 18 o 3

. . 12 oo o

E f:k O

0 0.4 0.8 1.2 1.6 '2.0 A C T I N O M Y C I N D C O N C . ( p g / m l )

Fro. 8. The effect of t rea tment with aetinomycin D upon the sensitivity lo RNase of radioactive material which has become associated with the polysomcs during a 10-rain pulse. The cultures were preincubated with aetinomyein D for 30 min before adminis t ra t ion of the "H-uridine. O, % of total polysomal radioactivity released by RNase; O, specific activity of isolated polysomes prior to RNase t rea tment (epm per mg polysomes • 10-3) .

L CELL POLYSOMES 55

synthesis of mRNA and rRNA, subsequent experiments utilizing different pulse labeling periods required that this interpretation be modified (Fig. 3). Hormone treatment causes a 15- to 20-min time lag in the entry of radioactive RNase-resistant RNA into the polysomes. Su- crose gradient analyses of polysomal RNA from hormone-treated and control cultures substanti- ate that this is due to a delay in the appearance of new rRNA (Fig. 10). The delay may be caused by a decrease in the forward rate of one or more steps of the ribosomal synthetic process: perhaps the synthesis or maturation of rRNA, the assembly of the ribosome, or the transport of the new ribosomal components to the cytoplasm.

Discuss ion

L cell polysomal RNA which is rapidly labeled with ~H-uridine was found to be primarily tlNase-sensitive (sen-RNA) while the RNA which becomes radioactive at a slower rate is more resistant to RNase (res-RNA). In growing cultures, labeling of the res-RNA and appear- anee of newly synthesized rRNA in the cyto-

TABLE 2 EFFECT OF HYDltOCOltTISONI.; UPON THE

LABELING OF POLYSOMES a

Pulse Amount of Labeling Isotope

Admin- Time istered

#Ci all- rain uridine/lO

ml medium

10 100.0 20 6.7 30 6.7

0.67 0.67

60 20.0 120 10.5.0

Average

Polysome Specific Activity

Hydro- Control cortisone

cpm/mg polysomes

36,560 18,390 4,780 1,510 9,840 4,370

605 336 762 381

129,000 46,200 162,000 43,400 74,000 30,500

Decrease

%

50 68 56 44 50 64 73 60

58

For each experiment two sets of 5-day-old cultures were refed with normal medium, with or without 10 -6 M hydrocortisone. Twenty-four hours later, these cultures were pulse-labeled with aH- uridine for the indicated length of time before harvesting. The polysomes were isolated, and their specific activity (cpm/mg polysomes) was measured as described in "Mater ials and Meth- ods."

b X

ILl e-

2

::D ~:n E 2

E

o 0

I l I I

(A) . , ~ -

e ~ ~

r I i I I

0

I

0

60 120 LENGTH OF LABELING

(minutes)

__ ! I ! I

i 2 o - (B) o

,lo / f o .c -

E

d o ' LENGTH OF LABELtNG

(minutes) Fzc,. 9. The effect of 24-hr exposure to 10-" .~

hydrocorlisone upon the uptake of 3H-uridine into the TCA-soluble fraction of L cells (A) 'md upon tim amount of ra(tioadivily associated with the TCA-insoluble fraction (B). The initial eoneenlra- lion of the labeled compound in the medium was 6.25 /~Ci per inl with a specific '~etivity of 21.7 Ci per mmole.

plasm follow similar kinetics. Moreover, synthesis of the rRNA and res-RNA are similarly inhibited by low concentrations of actinomycin D. Thus, there is reason to conclude that the newly synthesized radioactive res-RNA is ahnost entirely rRNA.

Identity of the more rapidly labeled sen-RNA is less readily established. Several facts, however, suggest that the major component of it is mRNA: (a) within the polysome structure there are regions of the mRNA between ribosomes which are particularly vulnerable to the action of pancreatic ribonuelease; (b) like mRNA the cytoplasmic sen-RNA is rapidly labeled to high specific activity by brief exposure of the cells to :'H-uridine; (c) low concentrations of aetinomy- tin D inhibit the labeling of sen-RNA only

56 RICHARDS, KITES, AND HERSH

i

LD 0

A NORMAL

c) "o

600 i

TOP

B HYDROCORTISONE TREATED

o ~ " - - - z ~ io TOO

Fro. 10. Sucrose gradient profiles of purified polysolnal RNA from cells labelled for 30 min wifl~ "H-uridine. - , opiieal density; "-', counts per minuie; A, control cells; B, cells exposed lo 10-" .~1 hydrocorfisone for 24 hr.

slightly but completely interrupt the labeling of rRNA. This differential effect of actinomycin D is consistent with known effects of the antibiotic on the synthescs of rRNA and mRNA (15-17). It should be mentioned that the syntheses of tRNA and 5 S RNA are not impaired by such low levels of actinomycin D (17); (d) tim amount of radioactivity associated with the small RNA species of the polysome is low com- pared to that associated with mRNA and rRNA. By process of elimination, therefore, the major component of sen-RNA must be mRNA. This diagnosis is in accord with similar evidence from rat liver in rive (25). As a corollary, the appearance of radioactivity in L cell polysomes after pulse labeling with "H-uridine may, as a first approximation, be considered in terms of the synthesis of mRNA and rRNA exclusively. Thus, digestion of polysome preparations with pancreatic RNase under the prescribed condi- tions offers a means of experimentally distin- guishing between newly synthesized mRNA and rRNA. Messenger RNA is released as low molec- ul'/r weight material while rRNA is not. The

exI)erimental distinction is not absolute since there is undoubtedly some cross-contamination between the two classes.

The effect of hydrocortisone upon the rate of appearance of newly synthesized rRNA in the cytoplasm was assessed by means of the technique described. The most obvious effect of the hormone is that it delays the maturation of rRNA in the process of its incorporation into cytoplasmic polysomes. Beyond this kinetic effect there is a large decrease in the extent of incorporation of the isotope into total RNA and polysomal RNA. Possible causes of the decrease include a lessening in the rate of RNA synthesis, an increase in the rate of RNA breakdown, and changes in the specific radioactivity of the RNA i)recursors. Since the amount of RNA present per mg of cell protein after 24 hr exposure to 10-" M hydrocortisone is slightly, but signifi- cantly, less than that in the controls, it is un- likely that changes in the specific activity of the RNA precursors c'm account for the entire effect, if indeed, an)-.

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2. Chen, H. W.. R. T. Hersh, and P. A. Kites. 1968. Environmcnlal effects on Ihe polysome content of arlificially cullured mouse cells. Exp. Cell Res. 52 : 490-498.

3. Drc~sden, M.. and M. B. Hoagland. 1965. Poly- ribo~onms from Escherichi.q cell: enzymatic method for isolation. Science 149: 647-649.

4. Staehelen. T., C. C. Brinton, F. D. Wettslein. and H. Nell. 1963. Structure and function of E. coli ergosomes. Nature (Lend.) 199: 865- 870.

5. Cox, R. A. 1969. The effect of pancreatic rit)onuc!ease on rabbit reticulocyte ribosomes and its interpretaiion in terms of ribosome structure. Biochem. J. 114: 753-767.

6. Sanfor(t, K. K., W. It. Earle, and G. C. Likely. 1948. Growth in vitro of single isolated tis- sue cells. J. Natl. Cancer Inst. 9 : 22,(t-246.

7. Lin. It. I., and O. A. Schjeide. 1969. Micro estimation of RNA by the cupric ion cata- lyzed oreinol reaction. Anal. Biochem. 27: 473-483.

8. Perry, R. P., and D. E. Kelley. 1968. Mes- senger RNA-protein complexes and newly synthesized ribosomal subunits: analysis of free particles and components of polyribosomes. J. Mol. Biol. 35 : 37-59.

9. Nell. H., and E. Stutz. 1968. The use of sodium

L CELL POLYSOMES 57

and lithium dodecyl sulfate in nucleic acid isolation. In: L. Grossman and K. Moldave (Eds.), Methods iu Enzymology. Vol. X I I B. Academic Press, New York, pp. 129-155.

10. Peterman, M. L., and A. Pavlovec. 1963. Ribo- nucleoprotein from a rat tumor, the Jensen sarcoma. J. Biol. Chem. 238 : 318-323.

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action of ribonuclease upon the polysomes of cultured mouse cells

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