Ultracentrifuge studies of RNA degradation

ARC’HIVES

OF

BIO(‘HEMISTRY

ANI)

BIOPHI-SICS

Ultracentrifuge JOSEPH

98,

214-223 (1962)

Studies HCPPERT

Received

of RNA Degradation AND JEAN

February

PELiUONT

13, 1962

Ultracentrifuge studies of RNA from Ehrlich ascites cells have shown that the original 28, 18, and 3-5 S componrnts change progrrssively as a result of drgradation. The 28 S component diminishes at a much more rapid rate than the 18 S, and their relative ratio changes as a function of the age of the RN;1 samples. Intermediary components of 24, 21, 15, 12, and 8 S with a transirnt existrnce have been observed during the degradation process. The similarity bet,ween spontaneous degradation and the effect of small quantities of RNasc (10m3pg./ml.) indicates that, this spontaneous degradation is chiefly due to the effect of traces of RNnsc not rrmorrd by the phenol extraction procedure. The possible relationship brtwccn the scyuenw of events during degradation. the macromolewlar fragments foi,metl, and the, struvturc of RNA are diwitswd. INTRODI-CTION

Instability of ribonucleic acid (RNA) preparations has been observed by all workers in their studies on the biophysical structure and biological activity of t,his nucleic acid. As far as we know, no report has been published on investigations into the sequence of even& involved in the degradation of an unstable RNA. During our comparative study by ultracent’rifugation of RNA extracted from animal and human tissues, whether normal 01 malignant, (I), we have noticed some diffcrences in the ultracent,rifuge diagrams of different samples of RNA isolated from the same tissue. In cert,ain of these preparations, complet,ely new components were seen. These irregularities could be best, explained by admitting that some kind of degradation of the RNA takes place either during the extraction procedure or during conservation. We therefore undertook a systematic ultraccntrifuge study of the changes occurring in RNA as a function of time and of the process of extraction. The results which will bc prcscntcd here will show the progressive appearance of transient subfractions which do not exist in 214

the original preparation of RNA. These subfractions seem to be chiefly due to the action of traces of ribonuclease (RNase) which remain in the preparations of RNA nft,er phcno1 ext’raction. M.ATERIAl,S

AND

METHODS

TUMORS Ehrlich ascites tiunor (nrnrly tctraploid) was maintainctl by intraperitonenl passages in Swiss mice 6-8 weeks old. Cells were harvested 8 days aft,cr inoculation with 10’ cells/mouse. Cells were washed three times with pllospllate-buffered saline and resuspended at 5 X 10’ cells/ml. in phosphate bnffcr 0.02 IQ’, pH 7.3, cthylenediaminc tetraacetate (EDTA) lo-” 111.They were either immediately extracted or stored frozm at -2O”C, until used. In the latter case, phenol was added before thawing.

RNA EXTRACTIOK (a) Standard

Pyepnm tion

To the cell suspension (at, room temp. 25°C.) was added an rqunl volume of water-saturated phenol (R. D. H. Analar) along with 10% (w/v) of glass beads (Ballotini Gi,ade 12). The mixture was shaken on the Mickle disintegrator for 10 min. at, mtuximum rate. For larger volumes. a magnetic stirrer turning at maximum speed was employed.

This apparently gnrc the same efficiency. After cenLrifugation of the susprnsion for 10 min. :lt 5000 r.p.rn., thr wat,cr phase was rcmovcd, fresh phr:nol was added, and the shaking procedure was rvI)eatc~d for 5 min. This was repcatrd once more. The last. water phaw was cwolcd in an iw bat1L. sodium notate was added to 2% final conccntration, antI the pH was adjwted to 4 with diluted acetic acid. Two \-olu~ncs of ice-cold ethanol wcrr lhe11 added. The RNA prcril)it:lte wV:tRccntrifugctl and washrd fire timrs with :I mislurc of 2 vol. clh:mol and 1 vol. of 0.14 ,II NaCI to wmo\.r all traws of phenol. This prwipitatc~ was dirsolvcd in phosphate buffer 0.02 :lII, pII 7.3, and distribut (vi into small tlibcs in such way it.5 to 11:lv(~ in cncll nrwssary for (~~1~ cslwrinlc,nt (2 tube thfx :irnomt mg. RNA). Tlrcl RX.\ in c;wh t ulw was rr1wccipitatrd with 2 vol. et,lknnol in thv 1,wS;cnw of 25 sodi~~in :Lwt:ktc~, :mtl stow1 as s~I(.I~ :11 4’ C. until rlatd.

In some ras~l, the RN.4 in solrltion was 11rccipitntcd by 1 .\I KaCl. Aftw 12-14 hr. at 0°C.. the precipitntc was collcctcd by crntrifuping, wwhcd with 1 JI NaCl. rcdiwolvrd in 1)hos~)hatC buffer 0.02 Al. and finally r(~l)l~cc.il)it:ltt~~l witlr ethanol as :h~\c.

(b) IlLI-d Prepred

RX.4 preparations made with one phenol estraction only, and was not detectable for all bcntonitet,reatcd 1nqx~i~ntion~.

RNAHE C’rystallinr~ Ixmcwatic RS;w (M;tnn Inc., \Vorthington 13iochemical Corp., Xutritional Biochemicals Corp.) was dissolvetl in 0.14 ‘$1 NUCI, 0.02 31 phosphate buffer. The stock solution was st,orrd in the frozen state. In two cases a chromatographic purification of RNasc on diethylnminoet hylccllr~lo~e (DILL) columns (6) was perfounrtl. Tllo fiwt czlution lwalc .I WI:: usecl. GLASSWBRE S~wi:~l viwc n-as takrn to avoid any accident:tl contamination vith RSasc. All glxswarc was first washed with chromic acid, lhoroughlp rinsed with distilled water, and finally SQtrrilized at 180°C. fol 30 min. All buffer solutions WXY~allt oclarl~c~l. The rdtrncentrifugc cells were clrnned with hot IiMnCL follow-cd by concentrated citric acid, and spwial rare was t akrn to avoid any handling with ihe

fir1gelY

(7).

with Bentonite

The swpcnsion of bcntonite (U.S.P. l;ishcr 1,aboratory Chemical) was prrparcd following the the method of Fracnl-xl-Conrat c’t r/l. (2). The nscites crlls, after washing with buffered saline were resuspended in distilled water to which bc~ntonite VW added to obtain a conccntrntion of 0.3-0.5 mg. dry wt./ml. The phenol extraction was donr as previously described rsccpt, that to rnch water l)hasc, fresh bentonite was added. The alrohol prrcipitate of RNA was dissolved in phosphatc, buffer with bcntonite, and the bcntonite was fin:rlly eliminat~cd by ccntrifnging for 1 hr. at 90,000 X g in tlw Spinco model I, pwpnrativc rentrifrp.

H S A was dctcrmincd by llr~ ldtrnviolot nbsorption qwctra (Spcctrophotometer Jol)in rt Tvon or lkkmnn) and by the orcinol renrtion (3). The ultwvioh~t absorption spectra for RiYl\ had a maxim11n1 at, 258 mu, a minimum at 230 mp and gave ratios for A25s/An~~between 1.9 and 2.2 and for AdA,:,, lwtween 1.8 and 2.3 according to the numbw of I)hcnol extract,ions. DN.4 was det,ermined by the Dische reaction (1). Only trares of DNA ~vcrc present Thv protein contrnt, as estimated by the method of I~~wry ct r/l. (5), was between 20 and 70 sg./ml. for the standard preparations. It was higher in

Sl)incx~ model E annlyt iv:d Illtrnwntrifuge v-it Ii schlicwn ol)tiw was used at bar angle 50” and sprcd 50.740 r.pm. Crntrifugntions wre carried omit brt\vorn 0 and 4’C. or at 2O”C., and ~~icturw wrrc Mimi (~\-rry 8 min. The tlrlralnmin crntct~l)iwe of 30 mm. was usually wed. In somP espcGncnta. the Kcl-F cell of 12 mm. w:~ rwd with itlrnticxl rrsults.

Figure 1 shows t,hc diagram of a standard preparation (optical density at 260 n+ = 30) centrifuged inmediately after dissolving the ethanol precipitate in phosphate Mfcr 0.02 111 at pH 7.3. Three components are seen: Two are fast’ nloving and in large quantity, the t’hirtl is in a niucli smaller amount and mow:: inorf slowly. A4dniitting that the linear extrapolation l/S as a funct,ion of the concentration is still valid in tlw high concentration range used, sedinlentntion coefficients of 28 and 18 S have km found in this case for the two nia,jor cowponcnts, and 3-4 S for the slowlg moving

216

HUPPERT

AND

PELMONT

left).

fractions. These results are in good agrecmerit wit’h those in the literature for RNA of ascites cells, calf thymus, and liver ribosomcs (g-12). The two fast components arc quantitatively precipitated after 12 hr. at 0°C. in the presence of 1 M NaCl; the 3-4 S fraction remains in solution under these conditions. The relative amounts of 28 to 18 S in the fraction precipit,ated with 1 M KaCl are evaluated by the method of areas corrected for radial dilutions. The ratio found for different preparations varies between 1.70 and I .8.5. The correction of ,Johnston Ogston for mixtures which eliminates the error due to the small arcumulation of the slow constituent’ at’ the front, of t,he rapid-moving boundary, has been neglect,ed. This effect would anyhow act in the opposite sense to t.he changes observed. SPONTANEOUS

DEGRADATION

OF

RNlZ

One st’andard preparation of RNA (in solution) was left at 20 C., and samples were taken and centrifuged after t,he following intervals of time: 30, 130, 230, 330, and 430 min., and after 24 hr. The diagrams of Fig. 2 show the progressive decrease of the peaks corresponding to 28 and 18 S very clearly from the 130 min. onward. The 28 X peak decreases more rap-

idly than the 18 S, and this is responsible for the progressive diminution of the ratio previously considerctl. After 430 min. this ratio aJ)proaclies t’lic value of 1. Figure 3a shows the relat,ionship of the area corresponding to the 28 and 18 S componcnts as a function of time. Figure 30 shows the ratio of the amount of 28 S component to 18 S component as a function of time. In the meantinre two intermediary components having sedimentation coefficients of 21 and 24 S appear between the two principal peaks. The 21 and 24 S fractions are hardly visible at 130 min. but are quite clearly seen at 330 and 430 min. The appearance of new molecular sizes, is also seen behind the 18 S peak, but in this case 15 S. It, was found that all these intermediary components have a very transient existence: they begin t,o climinish after 430 min., just when the original constituents themselves disappear completely. These intermediaries arc rcplaccd by constituents with sedimentation constants of lo-12 S which arc themselves replaced by products of 5-7 S, and finally by 3-4 X. After several days no sedimentable products can be observed. When 5 S particles are predomina~nt in the preparation, the fact that

217

the hypocliromic effect ncvcr cxcccds 2c; shows t,hat the degradation of RNA is accompanied by the liberation of large macromolecular fragments, indicating the rupture of only a small number of bonds. In fact, the degradation seems to follow a pre-rstablished plan, implying that the original structure is broken down into components which fall within a definite range of sedimentation coefficients and which are t~hemselvcs broken down further. The existence of intermediary peaks could be found in some preparat,ions on dissolving the precipitated RNA. This suggested that RNA may have undergone some changes during the extraction and in the course of preparation. To reduce as far as possible the

number of manipulations during the preparation of RSA, cells were broken anal shaken just once with phenol, and, after a brief centrifugation t.0 separate the two phases, the aqueous phase was examined clircctly on the analytical centrifuge without eliminating the phenol. Figure 4 shows the diagram obtained in the presence of 5% phenol. In the region of the meniscus, phenol causes the formation of a marked gradient which obstructs the observation of the slow fractions. The fast components give hypcrsharp peaks which have approximatively the same area as the normal peaks (especially as measured after slow diffusion). The ratio of the 28 and 18 S peaks under these conditions is 2.49.

218

HUPPERT

AND

One possible reason for this difference in the ratio of 28 S/l8 S for a standard (three phenols) preparation and for RNA prepared with one phenol could be the higher protein ta)

40 c A@ t

6 20

0

0 8

1

*

0 a 1.0

1.5&i Ii

A A

A A

A

0.51 0

1.0

content of the latter. This seems unlikely, since, as will be shown later, RNA prepared in the presence of bcntonite still gives the same high ratio even after three phenol cxtractions. Thus the ratio of 1.86 for 28 S/ 18 S for RNA prepared by the standard procedure would indicate t.hat some degradation is already taking place under such conditions. ST.~BILIT~

0 0

tb)

PELMOST

log t/t,

Fro. 3. (a) Plot of the areas c~ouwponding to the 28 S peak (black circles) and the 18 S peak (open circles) on ultracentrifuge dingl~am~ (rf Figs. 1 and 2) of RNA stored in sohltion at, 20°C. I’OI various times vs. log t/to where to = time at whirh the RNA was dissolved and t = storage time brforc centrifuging. (0) Plot of K VP. log f/lo where K = ;nw of the 28 S peak/aw;~ of the 18 S prak.

IX

THE

PRESESCE

0~

PHENOL

The finding that with schlicren optics an analysis can be performed in the prcscnce of phenol has offered the possibility of examining the effect of phenol on the stability of RNA. Indeed, RNA preparations containing 5% phenol arc effectively nmch more stable than standard preparations. After 48 hr. at 2O”C., thcrc arc only rather insignificant changes in t’he diagram After elimination of the phenol the same sequence of degradation stages, as a function of time, arc observctl as previously described for a standard preparation. On adding phenol once again to saturation or half-saturation, further degradat’ion id inhibited and the RN,4 retains the ch:lract,ci,i~tics ( intermediary peaks) which it poss~scd at thcb time of adding phenol. However, cvcn at saturation, inhibit,ion of degradation is never complete, and, if the time interval is long enough (scvera1 days), complctc degradation occurs.

Fro. 4. Ultracentrifuge picture of the first :rqurous phnsc during tllcx 11h~nol c~stwrtion oi RNA from Ehrlich awites cells A,,,,, (without phenol) = 45. Conclitions of rrntr,ifuging as described for Figs:. 1 and 2. The solution is sat,urated with phenol, whic.11 ww-cs :i marked gradient close to the meniscus. The 28 S yak on the left shows :I rn:wkvd self-Aarpening effert.

OTHER ATTEMPTS TO STABILIZE RNA

Another method of obtaining stable RNB has been suggested by the finding of Fraenkcl-Conrat et aZ. (21 ~110 ~l~oweti that tlic addition of bcntonite during the cxtraction of infectious RNA from tobacco mosaic virus (TP\IV) allot~etl an RSL1 to be 0l)tained which loses its infectious activiti much more slowly t,han normal prrparations. \Vlicn 0.3 mg. bcntonite was addetl for each milliliter of our st,andard RNA prcparations, the degradation was inhibited at that particular &age to which the RNA was already degraded. When bentonit’e was wtldcd to the first stage of extraction at which the cells are being broken up, the schlicrcn diagrams of the RNA were identical to t’hose described for RNA prepared by one 1)hcnol extraction and without’ removing the phenol (but, without, the disturbing gradient, due to the phenol) ; this remains unchanged after further phenol extractions. The removal of thtr hentonitc (low not, change the stability. On the other hand, no significant, results were obtained by the addition of EDT.4 to a final concentration of 0.01 M, or of the antioxidants like cysteine or hydroquinone to a concentration of 0.5 mg./ml. Acidification at pH 4 slowed the degradation process without completely climinat,ing it. Tentimes repeated phenol extractions at 20°C.. followed by precipitation IT-it1166% et,hanol and washing, does not improve the stability of RNA. 8tandard RNA preparations conserved as an alcohol precipitate at 4°C. retain t.he same sedimentation diagram after several weeks. ENZYMIC DEGRADATION OF RNA

The diagrams of degradation described above were compared with those obtained after addition of different amounts of pancreatic ribonuclease. The use of bentonite or of phenol permit,tcd the reaction to be stopped at any given time. Bentonite has the advantage over phenol in not introducing disturbing gradients in the ultracentrifuge since it is already sedimcnted during the acceleration period of the rotor. The addition of RNase in amounts vary-

RNase /%ll

- hhi

---I

4I-4

16” I 16” 4 I

Id’ l---1 -I Id

-4

Temp.

ing from lOPA to 10-l pg./ml.’ causes the appearance of the same diagrams (i.e., intermccliatr peaks) as those described for spontaneous degradation at the same temperaturc, hut, in a much shorter interval of time. Figure 5 sl~ows a schematic reprcscntation of the diagrams at the end of 30 min. at 0, 20, and 37°C. with different doses of enzyme. RNase at, lo-” ~g./ml. complctcly eliminates the 28 and 18 S components, and 10-12, 5-7, and 3-4 S appear as in the case of spontaneous degradation, but again in a niuch &ortcr period of time. The 24, 21, and 15 S components are hardly visible and can be correctly measurcd only lvhen the amount of RKasc is less than lo-:’ ~g.:‘iiil./mg. substrate. On the ‘Tile epccific actiritics of diffrwnt enzyme pwp-

220

HVPPERT

AND PELMONT

other hand, any concentration of enzyme grcatcr than 0.1 pg./ml. causes such a rapid degradation that no intermediary products can be detected. The action of RNasc described is accclerated by an increase of temperature. .4t 37”(‘., the activity of amounts of RNase was small as lo-;’ ~g./nil. can be detected by changes in the diagram produced after 30 min. incubation wit,h a RNA preparation treated by bentonite where the effect of spont,ancous degradation can be neglected. For low temperatures int,ermediate peaks can bc seen with a relatively large concentration of cnzymc ( lo-” pg.). The addition of phenol slows down the enzymic activity n-iGout completely suppressing it. An Ri’CA preparation at’ I mg./ ml. in the presence of 5% phenol and 0.1 pg. RNasc does not show any scdimentablc material after 18 hr. at 20°C. An KKA l)reparation with lo-:’ pg. RKase, shows no visible change after 30 min. If now the phenol is rcmored, the cnzymic degradation is restored immediately. This shops that the altered the phenol has not irreversibly RXase but simply sloww~ down its action. From a consideration of the analogies listed above, it looks most likely that’ what WC descrihcd as spontaneous degradation is really due to the action of residual RNasc of cellular origin, contained in the RX-4 preparation. The activit,y of this r&dual RI%nse can bc estimated as being cquiralcnt to 1OWj pg. RNasc/mg. RIVA if, really. all RSL4 degradation is of mzymic origin.

In the course of the experiments described, we have found for RNA of Ehrlich ascites cells, three classical components: 28, 18, and 3-5 8. The intermediary peaks that we found do not seem to be dependent on the actual centrifugation procedure as was observed in the case of Schumnker and Msrano with DNA (131 since (a1 the degradation to which wc rcfcr took place during storage am1 not in the ultracentrifuge cell, and stable RXA was not affected; Ch) the same type of diagrams wre obtained when a Kcl-F cell was used.

Therefore the finding that, when RNA is stored in solution, degradation takes place with the appearance of inacromolcculc:: with intcrmediatc scdimcntation cocfficicnts. gives rise to :itf least’ two problems: 1. What is the cause of this spontaneous degradation of RNA? 2. M’hnt is the possible relationship between the wqucnrc in which the ncwl) formed macromolecules appear and the initial structure of the RNA moltrule? 1. THE

CAUSE OF SPONTASEOVS ~)EGRADATION

From the very first account of infectious RIG! of tobacco mosaic virus FracnkelConrat and Singer ( 14) put forward the suggestion that the inactivation of the RNA preparat’ions may be due to contaminating cellular RNase. This same explanation was further invoked by lat.er workers to account for the instability of infcct,ious nucleic acids from different o&jns. In our work, spontaneous degradation and enzymic cligwtion both girr similar ultraccntrifuge patterns. Both react in the sanic way to several types of inhibitors of RNasc and respond in the same manner t,o changes in temperature. This st,rongly suggests that spont,aneous degradation is largely due to residual RNasc of cellular origin which ia incompletely eliminated during the extraction proccdurc. Nevertheless, the possibility of some nonenzymic hydrolysis which follows the same pattern cannot be excluded. In such a case the enzyme would serve simply as a catalyst to accelerate this hydrolytic process. Eigner et c/l. I 15) have calculated the rate of hydrolysis for the thermal degradation of RKA. Their ~1~s are much lowr than ours at all comparable tcmperaturcs. Our experiments show that the densturing action of phenol on RNase can only be partial! if any. In fact, RNase at conccntrwtions less than IO-’ pg.jmg. substrate is apinhibited by phenol. parently reversibly whereas liigtier concentrations of cnzymc continue their action eren in the prwncc of phenol at snt.uration. This result is in accordance with the findings of Bachrach ( 16), who observed that RNA4 cxtractetl from foot.-and-mouth

disease virus shows no infectious actiyit,y if RKase is added to the virus preparation before the extraction procedure. The decrease of the RNase activity in the presence of phenol is presumably due to the action of phenol on both the enzyme and substrate. In connection with this, it is interesting to note the results of Findlay ( 17) and Eliidi ( 18 I who have both shown that RSnse is still active in the prcsencc of certain organic solvent’s, In addit,ion, &mint et cd. ( 19j hare shown that salt at high concentrations can inhibit small amounts of RNase (
AI\;D STRUCTURE

The fact, that the degradation \Cth t,imc of our initial RNA preparations gives rise to a defined sequence of transient intermediate constituents with decreasing sedimentation rates shows that the hydrolysis follows a pre-established plan. The macromolecular fragments liberated are of a definite size and seem to correspond to many subunits. The 18 S component itself may be a subunit of the 28 S. The observation t,hat’ there is a more rapid decrease of the 28 S than the 18 S component, niay indicate that, the 28 S conlponcnt has a higher degradation rate than the 18 S; this in itself does not exclude t’lie possible replacement of the 18 S coniponent by the degradation of the 28 S. The existence of intermediate peaks of different sizes provides evidence that all suscept~ible points on the molecule are not, attacked simultaneously and that the hydrolysis is indeed not at. random. If random hydrolysis did occur, particles of all porsiblt

sizes would be produced and this would show up in the ultracentrifuge in the form of one broad boundary. The degradation through intermediate coniponcnta can be explained by se\-era1 hypothc~sea: ( (1I The first att’ack on the RX-4 inolcculc gives rise to relatively small subunits, which art> subsequently rcassociatcd by the iiictal ions presmt (22 ) ; (0 ) the cnzyine naturally present, or added, is a complcx of enzymes act,ing in sequence ; ( c) The enzyinc present is hoinogcneous but the substrate has preferential points for attachiuent of the cnzynic.

This scww unlikely since intermediate peaks IThich appear in the presence of EDTh 0.01 -11, arc similar to those svcn when the RNA solution contained lo-’ to lo--’ ~11Alg ions. In fact, the complete reversal of degradation sequence has never been observed. Tow cam of aggregation only llave been noticed: (a) nlg++ (1 M) precipitates RNA even degraded to the 3-5 S components ; ( b) 0.1 M nlg+ + or phenol addecl to RX-4 in the 3-5 S stage gave polydisperse diagrams with scdiincntation coefficient:: up to 7 S. HYPOTHESIS

Ibj

A4ccording to this hypothesis, RSase must contain at least, two components, one prcscnt in large amounts and capable of cutting tlic substrate into large inolecules corresponding to the intermediate peaks, and another present in a smaller amount which can only act on t,liese products resulting from the action of the first’ enzyme. The products of the second enzyme would be polynuclcotmidea of random iizc. So far no evidence has been produced for tlic cxistcnce of such a ron~l~lcx of enzymes for pancreatic RSase. The experimental results reported hcrc hnvc always been the same regardless of the origin of the RKase or whether the RKase preparations had been highly purified by I’Cpcatcd chromatography. -It’ any ratr. the existence of such a complex for RNwse would lcarl to the third hypothcsie at, least for the cmzyine which arts first.

222 HYPOTHESIS

(c j

The clxistence of some kind of hctcrogeneity on the RNA chain, which mav favor attack of RNase at preferential points, or may favor the breaking of the molcculr at’ definite sites after enzyme attachment must’ be considered. This last hypothesis can be fitted into the model proposed by Doty et nl. (23) for the secondary structure of RSA. In this model 6096 of the bases are in complementary hydrogen bonded pairs and the others form single-stranded loops. With such a folding of the molecule, it is possible that only a few sites are immediately accessible to the enzyme, while the other sites would become progressively available as the molecule rearranged itself following t.he first’ cnzymic attack. On the other hand, it could also bc possible t’hat RNase may be attached to the molecule at many sites simultaneously, but that the enzymic hydrolysis can only initiate a rupture of the molecule if the cutting took place either in a single strand outside of a loop or in a double-stranded fragment of the molecule, but, at the same level in each strand. The striking effect of the increase of temperature on the speed of the degradation would thus correspond to changes in the secondary st,ructure (24). At such tempcratures, or under conditions where most, of the hydrogen bonds are broken, no int’ermediary peaks should be found. The difficulty of observing t,he int’ermediary components even at low trmperatures, when relatively high concentrations of RNase are used, could be explained by supposing that under these conditions, scissions at the same level in double-stranded regions of the molecule can occur with higher frequency than in susccptiblc single strands. The detailed study of the kinetics of formation for intermediary components during the RNB degradation lvill perhaps provide an experimental approach for verification of tllct model for RNA proposed by Fresco et al. (25). The isolation and analysis of tliesc intermediary components should furthcr facilitate the study of the structure of RNA4.

In the light of our data, some contradictory evidence in tht> literature about the sizta of subunits can possibly be cxplaincd. L4lI thtl sizes of subunit)s described by dif?clcnt aut,hors ( 12, 22, 26, 27) havc~ been found in our experiments according to (a) the amount of Rn’asc used; (b 1 the temperature of the reaction; and ( CJ the time during which the reaction took place. It would therefore seem that the disparity in their results are due to t’lie experimental conditions employed, including the effect of addecl proteins or inorganic ions on the degradation process. From all these results, we can conclude that RNll is degraded into subunits which seem to be related to t,he secondary structure of the RX-4 molecule. This does not necessarily imply, however, the RNA as such is built up of subunit,s. A reply to this last question will be obtained when a specific high molecular weight RNA is built up from subunits so as to exclude random nonspecific aggregation. ACKSOWLEDGMEXTS The aut,hors acknowledge thr kind 11~11)of Mr. G. Quash in revising the manuscril)t ant1 rorrrct ing the English. WC thank also Miss M. M. Breugnon for her skilled assistance. REFERENCES 1. IA:OUR, F.. L.WOLX, J., HAREL, H., ASUDHUIWXT, J., J. Satl. Cancer Iust. 24, 301 (1960). H., kk~oER, H., AKD TSXITA. 2. FRAENKEL-CONRAT, A., T'idogy 14, 54 (1961). G., J. BkZ. Chem. 214, 59 (1955). 3. CERIOTTI, Z., Nihchemie 8, 4 (1930). 4. DISCHE, nT. J., FARR, .I. I,.. 5. LOWRY, 0. H., ROSEBROUGH, AND RANDALI,, R. J.. J. Rid. Chcm. 193, 265 (1951). 6. MAVER. M. E., PETERSON, EJ. A.. SOBER, H. A., AND GRECO, A\. E., Ann. A’. I’. Ad. Sci. 81, 599 (1959). J., AND FREED, R., Nature 190, 921 7. TABKHNKK, (1961). 8. SCH.4cHX4x, H. K., “Cltracentrifugation in Biochemistry.” :icaclcmic Press, New York, 11)59. F. s. x., BROW, R. :I.. COI,TER, J. S.. 9. TIJIASHRF, AND ~.4vI~:s, M., &x’ki~~. el &p/q/s. /lclrr 27,662 (1958). 10. VIZOSO, &I. D., AND ~u~iwss. -2. T. H., Nioc/icm. Rioplr ys. Rcsrwch Commons. 2, 102 (1960). 11. CImw. P. I-., Sniurc 184, 190 (1959). 12. Har., IS. D., AXI) DOTU, P., .I. .11d. Jli~~l. 1, 111 (1959).

13. SIXIJMAKER,

V. IX., .+sn MztRaro,

13.. dixi!.

Nin-

chem.Biophys.94,532

14. 15. 16. 17. 18. 19.

20.

(1961). FRAEXKEL-COKHAT, H., .IKD HISGER. 13.. H~tll. .soc. chim. biol. 40, 1717 (1958). EIGSER, J., BOEIITKER, H., AXU RIrc~r.~~.s, Ci.! Biochirn ct Bioph y.s. Actrr 51, 165 (1961). RACIIH.~(.II, H. L.. T-idogy 12, 258 (1960). l~rr~~r..\~, n., h’hTHI.\S, .I. P.. AXD RAMS, n. R.. :Vutrm 187 601 (1960). Ikiinr, I’., I?ikhirn. et Riophys. Acln 53, 218 (1961). $‘RUKT, li., ~
G.

DE,

dcarl.S&81,570

AKD

ALLARD.

(1959).

c.,

A/r/L. s. 1..

H., J. JIol. Ijiol. 2, 171 (1960). J. R., .\LRERTS. n. &I.. ASD hn, Ytrturrp 188,98 (1960).

24. BOEI)TKLX,

25.

~cRIM'O,

26.

THROWS,

R.

A.,

~XLLEM,

I<.

h.

o.,

ASD

I’..

COLTER.

J. S.! X:al tti’c 187, 509 (1960).

27.

0T.lK.4,

E.,

0OT.4.

191,598 (1961).

1-i.. ASI)

0s.~~.\.

8..

Xctlure