Recovery of Labeled Ribonucleic Acid Following ... · Recovery of Labeled Ribonucleic Acid Following Administration of LabeledAuxinto GreenPea Stem Sections' ... room temperature

Recovery of Labeled Ribonucleic Acid Following Administrationof Labeled Auxin to Green Pea Stem Sections'

F. E. Bendafia,2 A. W. Galston, R. Kaur-Sawhney, and P. J. PennyJosiah Willard Gibbs Research Laboratories,

Department of Biology, Yale University, New Haven, Connecticut

Introduction

Recent experinm,ents with vertebrates (14, 17. 21.36), insects (6, 10) and plants (2. 18. 19, 26, 28, 34)have shown that RNA and protein synthesis are in-volved in the action of a wide variety of hormones.In particular, the blockage of hormone action by lowconcentrations of the RNA polymerase inhibitor,actinomycin D, indicates a connection between hor-mone action and the de novo synthesis of nucleicacids. It thus appears that the problem of explain-ing the molecular mechanisms involved in initial hor-mone action could be attacked by a study of the de-tailed mechanisms connecting hormones with nucleicacid metabolism.

Previous work from this laboratory (12. 16) hasshown that a partial oxidation of the plalnt growthhormone indole-3-acetic acid (IAA) can complex invitro with RNA extracted fromii growinig pea stemsegments. This finding led us to inquire whether.similar complexes between auxin miietabolites alld niu-cleic acids could be formed in vivo.

Materials and Methods

Ten mm long subapical stem sections derived from14 or 15-day-old light grown pea seedlings, var.Alaska (11) were incubated in petri dishes underfluorescent light (ca. 500 ft-c) in 10 ml of solutioncontaining IAA, generally 10-4 M, 1 % sucrose and0.01 M K phosphate buffer, pH 6.0. This high con-centration of JAA, which would be supraoptimal andinhibitory if fed to the conventionally employed eti-olated sections, is on the ascending limb of the dose-response curve for green sections (13). It is thisfact, we believe, which renders our tracer experimentsboth technically feasible and physiologically mean-ingful. To work with stimulatory levels of IAA inetiolated tissues (i.e. ca. 10-6 M) would mean im-possibly low levels of radioactivity in isolated frac-tions. To work with high levels (10-4 M) of IAAwith etiolated tissue would mean injury and growthinhibition, rather than growth promotion.

1Received January 11, 1965.2 United States Public Health Service Postdoctoral

Fellow. Present address: Departamento de Biologia,Universidad Nacional de Nicaragua, Leon, Nicaragua,Central America.

After various time intervals ranging froml 1 to 18hours the sections were removed and washed. andtheir length and fresh weight measured. They werethen stored in a deep freeze prior to extraction ofRNA. To insure stability of RNA during the ex-traction procedure some investigators add ribonu-clease inhibitors, such as bentonite (18, 19). \Vefound no need to do so sinice exogenous RNA sup-plied to our homogenate fractions in the presence ofphenol could be quantitatively recovered. A furthercheck involved a direct determination of ribonucleaseactivity in the pea stem and in fractions thereof.Although we can readily detect ribonuclease activityin one 5-mm stem section, no activity could be de-tected in any of the fractions in the floxv sheet de-scribed. in which one hundred 10-mm sections werehomogenized in the presence of pheniol (20). Thefrozen plant material (ca. 3 g) was homogenized toa still-frozen slurrv witlh a prechilled mlortar andpestle in 2 volumiies of freshly redistilled phenol (90:10 with Tris. v/v) and an equal volune of 0.01 aiTris-HCl buffer. pH 8.0. The homogeniate was per-mitted to stand at room temperature for 1 hour, cen-trifuged at 4 + 10 for 20 minutes at 3500 X g, thephenol-water layer removed and extracted 4 X atroom temperature for 1 hour with 0.01 M Tris-HClbuffer, pH 8.0. The RNA in the combined aqueouslayers was precipitated by the addition of 2 %(final conc) potassium acetate and 2.5 to 3.0 volumesof cold ethanol (95 %). This minixture was allowedto stand at 4 ± 10 overnight. and the white flocculentprecipitate harvested by centrifuging the mixture at20,000 X g for 20 minutes in the cold. The pre-cipitate was dissolved in 2 ml of Tris-HCl buffer.pH 8.0, and centrifuged at 30.000 X g for 20 minutesto remove any debris. The supernatant fraction wasagain treated with 2 % potassium acetate and 2.5 to3.0 volumes of cold ethanol and allowed to stand 30minutes in an ice bath, after which the precipitatewas again harvested by centrifugation and redis-solved in 1 nil of Tris. This procedure was repeated3 more times to yield a purified RNA (Ppt IV),which had a constant spectrum and specific activity.Reextraction of this fraction with phenol changedneither its spectrum nor its radioactivity. All workreported here was performed on Ppt IV, whose iso-lation is su1mmlarized in figure 1.

Ribonucleic acid was hydrolyzed wvith 0.3 At KOIHat 370 according to the technique of Davidson and

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PLANT PHYSIOLOGY

EXTRACTION OF RNA FROM PEA STEM TISSUE

PEA STEM SECTIONS

FREEZEHOMOGEN!ZE IN 2 VOL/WT REDISTILLED PHENOLADD EQUAL VOL TRIS-CI BUFFER, 001 , pi 80CENTRIFUGE, 4±1°C

AQUEOUS + PHENOL LAYERS PLANT RESIDIJESHAKE HR, ROOCM TEMPERATURECENTRIFUGE, 4 ± 1°C

AQUEOUS LAYER PHENOL LAYERREPEAT TRIS EXTRACTION 3XCOMBINE AQUEOUS LAYERS

MAKE 2% WITHPOTASSIUM ACETATEADD 2.5 VOL COLD ETHANOLSTORE OVERNIGHT, 4 ± 1° CCENTRIFUGE

ters wsere used for measuremiienit of the radioactivityof the various fractionis.

Results

lIIcorporatioli of Label into RNA. The frac-tiOin designated as precipitate IN' ds fouund to havethe spectral characteristics of RNA (fig 2) and tobe completely hydrolyzable by crystalline ribonui-clease. WVhen extracted from steml tissues incubatedwith C'4-IAA, precipitate TV was also radioactive.'The specific activity of this fraction was found to beunaltered by dialysis in the presence of coldl IAA ancdhy extraction with suclh organic solvents as ethanol,ethyl ether, chloroform and to further l)henol extrac-tions. No DNA could be detected in precipitate IN'by the p-nitrophenylhydrazine mlethod of Webb and(

STANDARD PEA RNA ABSORPTION SPECTRUM9 n

INSOLUBLE RESIDUE (DISCARD)

REPEAT ETHANOL - K ACETATE PRECIPITATION

AND RESOLUTION IN TRIS 3 X.

PRECIPITATE |DENSITY(-GRADIENT

ELECTROPHORESIS CENTRIFUGATION

UV SPECTRUM RADIOACTIViTY HYDROLYSISMEASUREMENT

Fi(;. 1. Scheme for the isolation of pea RNA.

z

0

co

21

l o

081

061

04

021

Smellie (7) and the monoriboniucleotides separatedelectrophoretically by a slight modification of theMarkham and Smith technique (23). The bufferused was 0.05 M ammonium formate-formic acid, p1l3.4. containing 5 % sucrose to decrease trailing ofspots. The electrophoretic separation wvas per-

formed with a Spinco Duostat and Durrunm-type cell,with a voltage gradient of 9.8 v/cm11, applied for 4hours. The nucleotides were detected and markedunder a UV lamp; the spots were then eluted with0.01 N FICI, spectrally characterized ancl counted.Thin-layer chromatography was also used to separatethe nucleotides. The adsorbent w,vas MIN celluilosepowder (300Gs/DEAE cellulose) sold by Brink-mann, Inc. TI'he developer was .01 N HCl, as reconi-

iended l,y Raivierath (27). TI'he C14-IAA used inImlost of the experimiients was a carboxyl-labeled pro-

duct ( 16.9 irc/maI) synthesized by our colleague B. B.Stow-e *(31 ). B3oth -as floxw and scintillation cooIIn-

0 300 290 280 270 260 250 240 230

WAVE LENGTH (mp)FI(;. 2. Comparison of the spectral characteristics of

pea and yeast RNA. RNA was dissolved in 0.01 M Tris--HCI buffer, pH 8.0, and characterized in a model 350Perkin Elmer recordIiing spectrophotometer.

Levy (35), Ino P)rotein was detected by the Folinreagent (22) nor wxas any loss inI material or specificactivity found after treatment wvith 2-methoxyethanol,a mlaterial used byb Kirby (20) to remove polysac-charides. It was thus concluded that the radioac-tivitv found was an integral part of extracted RNA.The kinetics of inicorporation of label from carboxylC14-IAA into precipitate IN- are shown in figure 3.

The uptake of label from C14-IA..N into plant tis-stie rises as the concentration of IA\A is increase(d

SUPERNATANT FRACTION PRECIPITATE I

REDISSOLVE IN TRIS BUFFERCENTRIFUGE 20,000 X, 20 MIN

1.8

SUPERNATANT FRACTION

61

141

O.l mg/ml

X-WORTHINGTON YEAST RNA

/ tL* 257mp

_ ~ ~ ~ ~~~~~~~~~~_ ~/4

230mp

e.u

978S

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BENDANA ET AL.-RECOVERY OF LABELED RNA FROM LABELED IAA

z

DI, I I I I I

a:

2

0 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18TIME OF INCUBATION - HRS

FIG. 3. Kinetics of incorporation of radioactivityfrom IAA into RNA as a function of IAA concentration.

(table 1) and with increasing length of the incuba-tion period (table II). The data of table II showalso that under 5 % of the total counts supplied were

absorbed into the tissue during a 4-hour period, andthat, at any time chosen, under 1 % of the absorbedcounts are found in RNA. The remainder of thecounts must be located in other metabolites of IAA,

Table II. Kinetics of Uptake of Label fromInitial IAA was 6 X 10-5 M; specific activity 16.9 m(

Table I. Relation Betweent Concentration of AppliedCarboxvl-Labeled IAA (13.5 mc/mm) anid Labeling ofExtracted RNA (Precipitate IV).

Incubation time = 4 hours.

% Increase Cpm perIAA (M) in fr wt ag RNA

Control10-610,-10-410-3

15.430.846.261.673.0

0

0.481.196.18

27.6

not further inivestigated here. Such metabolitesmust include CO, or other volatile products whichare not accounted for in sections or solution.

To investigate the specificity of IAA and of thecarboxyl group as donors of label to RNA, methyl-ene-labeled IAA and carboxyl- and methylene-labeled 2,4-dichlorophenoxyacetic acid (2, 4-D1), were

fed to tissue and the resulting RNA isolated andcounted. 'rable III shows that IAA is approxim-ately 10 times more effective as a label donor thanis 2,4-D, and that in each compound, carboxyl is 2to 3 times more effective than methylene. This na-

turally raised the question of possible dismutation ofthe labeled compounds to smaller fragments, whichcould then be recycled, either photosyntheticallv or

heterotrophically, to yield components of RNA. Forcomparison, therefore, labeled CO. was fed, in thepresence of unlabeled 10-4 M IAA, to insure equalityof the growth response. Like C14-IAA, C1402 isincorporated into RNA. but the pattern of labeling

IAA into Sections and Incorporation into RNAC/mM. 100 sections per treatment.

Incubation mediumTotal

Time of Total % of Cpm as corrected % oftreatment Cpm per corrected counts IAA in cpm as original IAA

(hr) 4 ul cpm X 106 remaining 4 ,* IAAXX 106 rema:n ng

0 10,040 25.1 100 ... 25.1 100V4 8617 21.54 85.81 8880 22.20 88.44M2 8633 21.58 85.97 8544 21.36 85.091 8412 21.03 83.78 8202 20.50 81.672 8263 20.65 82.27 6873 17.17 68.404 7545 18.86 75.13 5115 12.78 50.91

Sections RNA (precipitate IV) Cpm illTime of Cpm Total cpm % RNA/cpmtreatment in 2 in all of counts absorbed

(hr) sections sections absorbed mg RNA cpm/,ug RNA X 100

0 ... ... ... 3.024 1734.9 86,745 0.345 3.21V2 3526.3 176,315 0.702 3.74 0.3 0.6361 9076.1 453,805 1.807 3.59 0.7 0.5532 11,770 588,100 2.344 3.50 1.36 0.8084 23,188.3 1,159,415 4.619 3.48 3.10 0.930

* 4 IAI spotted on TLC and only cpm in RF of IAA used.

979



Table III. Comparative Effectiv'encss of Differcntiall/v Labeled Auxins as Doniors of Label to RNASections were incubated for 18 hours before RNA wxas extracted. Growth, asimeasure(d b- the increase in fresh

wneight, was approximately equal in all 4 groups. All auxins supplied at 10-4 3M.

CpI)1Specific Activity a(lded

,uc/mg >< 10'

96.6 36.916.2 6.28.9 4.319.2 9.4

Totalcpm inRNAfraction

150,52010,6402,240640

TotalRNA( 111,g)

3.1502.9003.3352.875

Correctionfactor

cpm/,lg IAA-RNA 1(1= 1

47.783.660.670.22

5.958.583.29

from the 2 sources is quite differenit. RNA ob-tained fronm sectiQns fed IAA show a great pre-ponderance of label in the adenylate anid cytidylateregions, while RNA from sections fed CO2 show amiiuch more symmetrical distributioln of label in the4 nucleotides. The incorporation of label from bothIAA an(l CO, was nmarkedly inhibited by 10 ,ug/mlactinomycin D (table IV), while the incorporationof label from C1402 was ulnaffected by the pres-ence of IAA and therefore of growth. It shouldbe noted that auxin-induced growth is totally inhibitedby the 10 ,ug/ml actinomycin D (AMID) treatment.That some incorporation of C'-402 into R-NA shouldbe observed even in the presence of AM1D is not uln-usual, since it is well knowln (24, 32) that polylphos-phorylase, the enzyme responsible for the additioni ofthe terminal trinucleotide to s-RNA, is not inhibitedby AMID. Although this reasoninig nmay also he usedto explain some of counts found in the C'4-IAA fedsections, approximately 18 cpm/,ug RNA cannot beexplained in this wsay (table IV). These data areinterpreted as meaning that some of the counts go-ing from fed IAA to extracted RNA arise fromii re-cycling of CO, or some other fragment of IAA, but

Table IV. Effect of Actinonicin D (AMD) on Grow,thof Green Pea Ste1in Sections and Labeling of RNA.The sections were grown for 18 hours. IAA con-

centration 10-4AI; ANID concentration 10 gg/ml.*

Tre,

I AA

Labeled TLabeled

... L

...LlUnlabeled I

Unlabeled T

atment %Increase

co2 AMD in fr wt

.bInlabeled_abeled_abeledLabeledFabeled

35.4122.0

-4- 38.421.3

-1 11.3-- 113.0+ 33.6

Inhibi-tion(cpm/jg

RNA)Cpm/,ug due toRNA A-MD

30.624.612.56.7

11.35.5

6.00

5.8

5.8

* This concentration of AMD inhibits all auxin inducedgrowth measured as percentage increase in freshweight.

that the remainder of the incorporationi occurs 1vway of a mlore direct pathway, such as direct cou-

pling of IAA to preexisting RNA.Isolation of IAA froi the Labeled RAA. To in-

vestigate the possibility that IAA imiight also bindper se to RNA in a form recoverable from the com-plex, a sanmple of labeled Ppt IV was hydrolyzed withKOH, as described above, and also by the Na2CO,method devised by Zamecnik et al. (37). After hb-drolysis with KOH, the material was acidified to pH2.8 to 3.0 with 0.01 N HCl and rapi(lly extracted 3X with l)eroxide-free ethyl ether. \\thile pure IAApartitioned 2.5 % into the aqueous layer anid 97.5 %into the organic layer, the radioactivityof the com-

plex p)artitionedl 65 % into the a(qtneonls layer andl35 %, inito the ethereal layer. This indicates that thelabel now exists in several different molecutlar species.The ethereal layer xx-as takeni do -n to drvyness in a

rotary flash evaporator at room temperature. Ther-esi(llte was takeln up in 0.5 ml of absolute ethanol andseparated by thin layer chromatography oni silica gel,the Zolvent svstem being methyl acetate-isopropanol-25 % amnlmoiniuinm hydroxide (43 :33 :20, v/v/v). Thegel from indicated areas of the plates \vas scraped

off ain(I subjected to liquid scintillation counting ac-

cording to the techllique of Snyder and Stephens(30). Under these conditionis, where the RF of TAAis 0.47 to 0.50. the maajor peak of radioactivity ofthe recoxered silica gel occtirredl at the same position(fig 4). In another experiiient, spraying the tlhinlayer plate wvith the Prochazka reagent (217) pro-duced a spot at the locuis of IAA wxhich xvas readilydetected under an ultraviolet light and had an ap-pearance identical with similarl -treated authenticJAA. A p)ositive Prothazka reaction at the locus ofIAA was also found in unextracted alkaline hvdroly-zates of the RNA and in ether extracts thereof.Control RN'A, or RN.\N\ fromii C'40., fed sectioInsvielded no such spot or couInts ill their hvdrolyzateswhen chromatogramrmed. These results indlicate thatIAA or some metabolite of TAA comlplexes xvith a

RN-A fraction during growth of pea stemii tissue.Phy,sical CharacterAv-ation of the Labeled RAA.

Labeled RNA derived fromi sections fed carboxvl-labeled TAA wvas separated itn a stilcOse (lensitv -ra-

dient (0-25 %) according to the method of Brakke

Source(of label

IAA-C14JAA-2C'42,4-D-1C;42,4-D-2C"4

CorrectedCplln/ygRNA

47.7821.825.770.73

Relativecpnllmpg

100.0045.6612.071.52

98X0 PLAN-T PIIYS10LOGY




Unknown' 1~~~~~~~~.1p0.01 pg0

0.4 0.5 0.6 0.7 0.8 0.9 1.0

Rf

FIG. 4. Recovery of IAA from hydrolyzed RNA asshown by thin-layer chromotography on silica gel.Graphic representation of the average of 5 experiments.The pattern was obtained by removing the gel and deter-mining the radioactivity at each RF by liquid scintill-ation counting. IAA: specific activity 13.5 mc/mM; 170cpm/mpg IAA; average cpm at RF 0.5 was 250 cpm,or about 1.46 m,g IAA (8.3 X 10-10 moles). RNA con-tent per spot applied = 30 jug (average M.W.4s RNA =

30,000, about 80 nucleotide residues) or about 10-9 M.

RNA: IAA counts ratio = (10-9)/(8.3 X 1°-10) =

1.20. Therefore we have found approximately 1.0 moleof IAA per mole 4s RNA or one IAA per 80 nucleotideresidues.

(4), using a Spinco model L ultracentrifuge and an

SW25.1 rotor, at 24,000 rpm for 8 hours, in 0.01 M

Tris-HCl buffer, pH 8.0 and 0.001 M MgCl,. Fromthe very first detection of labeled RNA, the 4S frac-tion was most heavily labeled, and even after 18hours of incubation in labeled IAA, the 16S and 28Sribosomal peaks were unlabeled (fig 5). There is

I.8 -A9SORBANCY 260

i 6 0-RADIOACTIVITY0

1.4 -/ \1220

06

1.2 18 0

0.8-014

FR0.8AC\T/ION\N M0.6-i

0.4-60

0.2 02/

1 2 4 6 8 10 1214 16 1820 222426

FRACTION NUMBER

FIG. 5. Centrifugal profile of pea RNA in a 0 to25 % sucrose density gradient. One ml fractions were

collected and a complete absorption spectrum obtainedfor each. From right to left, the 3 absorbancy peaksrepresent 4S, 16S and 28S fractions. These figures were

obtained by comparison with known rat liver RNA.

also a peak of labeling, but not of absorbancy, in theregion between 4S and 16S, usually designated asmessenger RNA. This peak would appear to havevery high specific activity.

The question arises, is IAA iniitially incorporatedinto a 4S RNA fraction, or is it first incorporatedinto a heavier fraction, which is then broken downto 4S size? This was investigated by comparing thecentrifugal distribution of radioactivity in labeledRNA produced from simultaneously applied C14-IAA(13.5 inc/mM; 7.5 ,c added) and H3-uridine (4.6c/mM; 100 juc added). RNA was obtained and sep-arated centrifugally as previously described. A 2-channel scintillation spectrometer (ANS, Inc., Wall-ingford, Connecticut) permitted simultaneous deter-mination of H3 and C14 in each centrifugal fraction(fig 6). The counting efficiencies were as follows:in the H3-channel, 15.7 % with a 2.64 % carryoverfrom C14; in the C14 channel 61.34 % with a carry-over of less than 0.09 % from H3. All plotted valueswere corrected for background, carryover andquenching.

2.4

20

E 1.60

0

N

.2

co

CDi

0.4

0Q2

0- ABSORBANCY

O---O H3A-A C14

7''Ar '

4, .-,Y.

4s,.0

o-OO

i6s I

IA

.I

i I

IcI e_ v,

240 o10 12 4 go i 20 22 24 26 27

FRACTION NUMBER

120

-

I- °u ti-

_ 55 t- 44:

so1-

-40 t-35 ,* - 280 'O

- so E - E

-25 ') 200

IS20 52

L

ko§d. L Bikgd.

FIG. 6. Centrifugal profile and labeling pattern ofpea stem RNA, obtained from sections simultaneously in-cubated for 6 hours with 100 ,uc H3-uridine (specific ac-

tivity 4.63 c/mM) and 1-C14-IAA, (specific activity 13.5mc/mM). Sucrose gradient 0 to 25 % in Tris-HCl,(0.01 M pH 8.0, 10-3 M Mg,Cl. *----@, absorbancyunits; O----0, corrected cpm of tritium; A----A, cor-

rected cpm of carbon-14. Uridine is incorporated intoall types of RNA while C14 from IAA is associatedmainly with the lighter fractions even after this long in-cubation period.

It is obvious that tritiated uridine finds its way

into all RNA fractions after a 6-hour incubationperiod. However, C14 from IAA, as previouslyfound, is associated mainly with the 4S peak, sec-

ondarily with a peak in the usual vicinity of mes-

senger RNA, and practically not at all with the 28Sribosomal peak. In the 28S peak, the molar ratioof uridine to IAA incorporation was approximately

981

EC.V

Pu reIAA

0 0.1 0.2 0.3

250

200

15 0

100

501



PLANT PHYSIOLOGY

8.0. Hlowvever, in the 4S aid(I 16S regions the cor-responldinig figure wvas less thain 0.6. This substan-tiates the view! that IAA is preferentially incorpor-ated into liglht RNA fractiois, anid speaks against thelarge-scale ulnslecific incorporation of (legradationprodltlcts of L\A into general RNA synthesis.

Discussion

T'here are now sev-eral reports in tlle literatturedescribling the binding of steroidl lhormonies (25, 29,33) alnd carciniogenic hvdrocarl)ons (3, 8) to niutcleicaclids. In addlitionl, there are reports that synthetic)lant growth stubstances such as maleic hvcdrazide(5) and(i kiiiius (9) may be incorporated inito titicleicaci(ds, xvlerc they substitnite respecti-ely, for lnormlalpyrinildines anid purinies. Tlfhe l)resellt rel)ort of thebindinog of I.-A with R\NA, while it is the first indi-cation of comiiplex formationi betweeni a native plantgrowth hormiiolle allnd nticleic aci(l. falls into an estab-lished framiexvork. It is also instructive to note thatmitonmvcin anid porfiromycini are believed to bind tonticleic acid only after re(tictionl to ani aromiiatic in-dole (15).

It is clear that carboxvl-labeled IAA (lonates labelto the isolated precipitate I V in 2 ways: A) directbilnding, of initact IAA with RKNA, from w\hich form itis recoveral)le as sucli an(l [B ) dismiiutation, probablyinto CO.,, followN-ed by retitili cation of the split prod-ucts in de novo purine all(l pyrimidine biosynthesis.Neither the nature of the linkage between RSNA -n(lIAAinor the reasoni for the relatively heavy labelingof cvti(ldlate aln(l adenv late regionis are clear. In viewxof the known turnover of cvtidvy-late and(I adenvlateat the aminio acid acceptor ell(l of tralnsfer RNA (1)it is temptilng to postulate ani attachmenit of TAA atthis point. \VNe hlope to obtain ftirtlher iniformationrelative to this possibility.

If a comiiplex between I.\A alnd RN.\ does occturin vivo, -hat mnay be its significance and role ingrowth lpromlotion ? There are 3 obvious p)ossibil-ities: A) 'I'he hormnonie, thlrotLgh1 comiiplex formationiwith RNA, could be involved in remloving it fromii thesurface of another molecule, such as template DNAor ribosomlal RNA. We have as vet lno evidencerelevanit to this possibility. B) The formiiationi of thehormone-RNA complex coul(d confer tipon the RNAgreater stability tow-ard (legracldative agents. such asribonuclease. In sup)ort of this possibility, we havein fact already observed l)rogressively slower ribo-nuclease action on R_NA isolated from stemii sectionsincubate(d for increasiing periods Awith 1 - M IAA.These resuilts will be reported later in (letail. C )The possibility also exists that the IA\A molectule, orportion thereof, may possess informational valuewhen attache(d to a partictilar locus in an RNAchain. The general coniformiiational similarity be-tween the indole nucleus alndl the purine lncletus makesthis conjecture at least a possibility w^orth entertain-ing.

Summary

C14 fromii carboxyl-labeled indole-3-acetic acid(IAA) fed to excised, grownijg greeln pea stem sec-tiolns is progressively incorporate(l ilnto RNA ex-tractable by phenol. The kinetics of inlcorporationiresemible the kinietics of groxx-th ind(uIictioln b ITAA.(94 fromii methvlene-labeled IAA and(I from carboxvland mi,ethvlene-labeled 2,44dichlorophenoxvacetic acidwxere also incorl)orated ilnto RNA, but xwith low-er ef-ficienicv than froml carboxvl-labeled fAA. BFothgroxvth and C'4 incorl)oration inlto R'NA NvAere in-Iiibited by- 1() ,u-lnil actinomivcin 1). 'I'lTe label w-asincorl)oratedl into light (4,S) RNA fraction asshovl b)v- sucrose (lensitv gra(lienlt cenitrifugationi.vhile tritiatecl tirdlidie simultaneously applied wvas in-corl)orated ilnto ribosomial peaks as xvell. Alkaliniehydrolysis, follow-ed by separationi of the nicleotidlesby paper electroplhoresis and thini layer chromatog-raphv shoNved that the bulk of the C14 label is associ-ate(l Nvith adenvlate and cytidylate regions. Some ac-tivitv fromii fed labeled IAA wxas also detectetI in theformii of u-nchaniged JA\.A recovered froml the alkalilnehydrolvzate. It is collultided that somiie IAA couplesper se xvith soltible RNA while somne of it is degraded.and the split l)ro(ltdcts incltudinig Co., are uised in re-synthesis of R\.NA components.

Acknowledgments

Supported by granits from the National Science Founi-dationi, United States Public Health Service and theHerman Frasclh Foundatioin. The senior author is apostdoctoral felloxv of the USPHS. We wislh to expressour thanks to our colleagues Professor G. R. Wyatt forhis generous shariig of his scintillation counter, Pro-fessor B. B. Stowe for a gift of carboxvl-labeled IA.antd Dr. G. Braxvcrmian for advice. An oral report Onlthis wvork wvas presented in August 1964 at the XtlhIniternational Botanical Congress, Edinburgh and at theAmiierican Institute of Biological Scienice, Boulder. Anabstract appears in Plant Phvsiology 39: xxx, 1964.

Literature Cited

1. BERG, P. 1961. Specificity in protein synthesis.AninI. Rev. Biochem. 30: 293-324.

2. BISWA.\S, B. B. AND S. P. SEN. 1959. Relationshiphetxveen auxinis and nucleic acid synthesis incoleoptile tissues. Nature 183: 1824-25.

3. BOYLAND, E. AND B. GREEN. 1963. The effect ofpolycyclic hydrocarbons oln the thermal denatura-tion of deoxyribonucleic acid. Biochem. J. 87:14P-15P.

4. BRAKKF, M!r. K. 1953. Zonal separations by den-sity-gradient centrifugation. Arch. Biochem.Biophys. 45: 275-90.

5. CALLAGHAN, J. J., M. D. APPLETON, W. HAAB, ANDC. P. PORTANOVA. 1962. Incorporation of C'4-labeled maleic hydrazide by RNA derived fromS'accharomyrccs rcvr.isiae. Proc. Penn. Acad. Sci.36: 91-95.

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6. CLEVER, U. 1964. Actinomycin and puromycin:Effects on sequential gene activation by ecdysone.Science 146: 794-95.

7. DANVIDSON, J. N. AND R. M. S. SMELLIE. 1952.Phosphorous compounds in the cell. 2. The sep-

aration by ionophoresis on paper of the consti-tuent nucleotides of ribonucleic acid. Biochem.J. 52: 594-99.

8. DEMAEYER, E. AND J. DE MAEYER-GUIGNARD. 1964.Effects of polycyclic aromatic carcinogens on viralreplication: Similiarity to actino.nycin D. Sci-ence 146: 650-51.

9. Fox, J. 1964. Incorporation of kinins into theRNA of plant tissue cultures. Plant Physiol. 39:xxxi.

10. GALL, J. G. AND H. G. CALLAN. 1962. H3 Uridineincorporation in lampbrush chromosomes. Proc.Natl. Acad. Sci. 48: 562-70.

11. GALSTON, A. W. AND R. S. BAKER. 1951. Studieson the physiology of light action. IV. Light en-

hancement of auxin-induced growth in green peas.

Plant Physiol. 26: 311-17.12. GALSTON, A. W., P. JACKSON, R. KAUR-SAWHNEY,

N. P. KEFFORD, AND W. J. MEUDT. 1964. Inter-actionis of auxins with macromolecular constituenitsof pea seedlings. In: Regulateurs Naturels de laCroissance Vegetale. Editions du Centre Nat].Res. Sci., Paris. p 251-64.

13. GAI.STON, A. W. AND R. KAUR. 1961. Compara-tive studies on the growth and light sensitivity ofgreen and etiolated pea stem sections. In: Lightand Life. W. D. McElroy and B. Glass, eds.Johns Hopkins press. p 687-705.

14. HANCOCK, R. L., R. F. ZELIS, M. S1IAW, AND H. G.WILLIAMS-ASHMAN. 1962. Incorporation of ri-bonucleoside triphosphates into ribonucleic acid bynuclei of the prostate gland. Biochim. Biophys.Acta 55: 257-60.

15. IYER, V. N. AND W. SZYBALSKI. 1964. Mitomycinand porfiromycin: Chemical mechanism of activa-tion and cross-linking of DNA. Science 145: 55-58.

16. KEFFORD, N. P., R. KAUR-SAWHNEY, AND A. W.GALSTON. 1963. Formation of a complex betweena derivative of the plant hormone indoleacetic acidand ribonucleic acid from pea seedlings. ActaChem. Scand. 17 Suppl. 1: 313-18.

17. KENNEY, F. T. AND F. J. KULL. 1963. Hydro-cortisone-stimulated synthesis of nuclear RNA inenzyme induction. Proc. Natl. Acad. Sci. 50:493-99.

18. KEY, J. L. 1964. RNA and protein synthesis as

essential processes for cell elongation. PlantPhysiol. 39: 365-70.

19. KEY, J. L. AND J. C. SHANNON. 1964. Enhan1ce-menit by auxin of ribonucleic acid synthesis in ex-

cised hypocotyl tissue. Plant Physiol. 39: 360-64.20. KIRBY, K. S. 1956. A new method for the isola-

tion of ribonucleic acids from manmmiialian tissue.Biochem. J. 64: 405-08.

21. LIAO, S. AND H. G. WILLIAMS ASHAIAN. 1962.An effect of testosterone on amino acid incorpora-tion by prostatic ribonucleoprotein particles. Proc.Natl. Acad. Sci. U. S. 48: 1956-64.

22. LOWRY, 0. H., N. J. ROSENBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Proteini measuremenitwith the Folin phenol reagent. J. B-ol. Chem.193: 265-73.

23. MARKHAM, R. AND J. D. SMITH. 1962. The struc-ture of ribonucleic acids. 2. The smaller productsof ribonuclease digestion. Biochem. J. 52: 558-65.

24. MERITS, I. 1963. Actinomycin inhibition of RNAsynthesis in rat liver. Biochem. Biophys. Res.Commun. 10: 254-59.

25. MUNCK, A., J. F. SCOTT, AND L. L. ENGEL. 1957.The interaction of steroid hormones and coenzymecomponents. Biochim. Biophys. Acta 26:397-407.

26. NOODEN, L. D. AND K. V. THIMANN. 1963. Evi-dence for a requirement for protein synthesis forauxin-induced cell enlargement. Proc. Natl. Acad.Sci. U. S. 50: 194-200.

27. RANDERATH, K. 1963. Thin-layer Chromatogra-phy. Academic Press, New York.

28. ROYCHOUTDHURY, R. AND S. P. SEN. 1964. Stu-dies on the mechanism of auxin action: auxinregulation of nucleic acid metabolism in pea inter-nodes and coconut milk nuclei. Physiol. Plan-tarum 17: 352-62.

29. SCOTT, J. F. AND L. L. ENGEL. 1957. Molecularinteraction between purines and steroids. Biochim.Biophys. Acta 23: 665-67.

30. SNYDER, F. AND N. STEPHENS. 1962. Quantita-tive carbon-14 anid tritium assay of thin-layerchromatography plates. Anal. Biochem. 4: 128-31.

31. STOWE, B. B. 1963. Synthesis of high specific ac-tivity C14 carboxyl indoleacetic acid and of C14-nitrile indoleacetonitrile. Anal. Biochem. 5: 107-15.

32. TAMAOKI, T. AND G. C. MUELLER. 1962. Syn-thesis of nuclear and cytoplasmic RNA of Helacells. Biochem. Biophys. Res. Commun. 9: 451-54.

33. Tso, P. 0. AND PoNzY Lu. 1964. Interaction ofnucleic acids. I. Physical binding of thymine,adenine, steroids and aromatic hydrocarbons tonucleic acids. Proc. Natl. Acad. Sci. U. S. 51:17-24.

34. VARNER, J. E. 1964. Gibberellic acid controlledsynthesis of cx-amylase in barley endosperm.Plant Physiol. 39: 413-15.

35. WEBB, J. M. AND H. B. LEVY. 1955. A sensitivemethod for the determination of deoxyribonucleicacid in tissues and microorganisms. J. Biol.Chem. 213: 107-17.

36. WICKS, W. D. AND F. T. KENNEY. 1964. RNAsynithesis in rat seminal vesicles: stimulation bytestosterone. Science 144: 1346-47.

37. ZAMECNIK, P. C., M. L. STEPHENSON, AND J. F.Sco-rr. 1960. Partial purification of solubleRNA. Proc. Natl. Acad. Sci. U. S. 46: 811-22.

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Recovery of Labeled Ribonucleic Acid Following ... · Recovery of Labeled Ribonucleic Acid Following Administration of LabeledAuxinto GreenPea Stem Sections' ... room temperature