AIn2+-(NH4)2SO4-activated (NH4)2SO4-activated(NH4)2SO4-activated reaction represents extranucleolar or chromosomal RNAsyn-thesis. Materials and Methods.-Isolation of nuclei and assay

THE INTRANUCLEAR LOCALIZATION OF TWODNA-DEPENDENT RNA POLYMERASE ACTIVITIES*

BY GERD G. iIAUL AND TERRELL H. HAMILTON

DEPARTMENT OF ZOOLOGY, UNIVERSITY OF TEXAS, AUSTIN

Comnmunicated by T. S. Painter, March 17, 19067

DNA-dependent RNA polymerase of mammalian cells has been described byWeiss,' Huang, M\Iaheshwari, and Bonner,2 Goldberg,3 and Biswas and Abrams.4The activity of RNA polymerase is enhanced if detergents are added to the incuba-tion medium." I Goldberg3 found that ammonium salts at high ionic concentra-tions are particularly effective in stimulating the activity of the polymerase.

Widnell and Tata" 6 have described two different RNA polymerase reactions forisolated whole nuclei of rat liver: the first manifests when nuclei are incubated inthe presence of M\IgI+ and the absence of ammonium sulfate; the other when nucleiare incubated in the presence of j\IJi2+ and the salt at high ionic concentration. Interms of base composition and nearest-neighbor frequency, the product of the.Mg2+-activated RNA polymerase reaction is a ribosomal type of RNA, whereasthat of the AIn2+-(NH4)2SO4-activated RNA polymerase reaction is a more DNA-like RNA.6 The mechanism of the action of ammonium sulfate in inducing RNAsynthesis is uncertain. Conceivably, the salt could enhance template activity as aresult of removal of histones7 8 or other DNA-associated protein, or the activity ofthe polymerase enzyme(s might be affected.5' 6A variety of investigations have shown that ribosomal RNA is synthesized in the

nucleolus9-1" (for review see ref. 12). Furthermore, DNA-dependent RNA polym-erase has been shown to be present in isolated nucleoli'3 and to possess a highturnover rate.'2 In the light of this knowledge and following the suggestion ofWidnell and Tata,6 we undertook to determine whether the \Ig2+-activated RNApolymerase reaction represents nucleolar RNA synthesis, and whether the nIn+-(NH4)2SO4-activated reaction represents extranucleolar or chromosomal RNA syn-thesis.

Materials and Methods.-Isolation of nuclei and assay of R.NA polymerase: From the liver ofadult female Sprague-Dawley rats, nuclei were isolated, purified, and assayed in vitro for RNApolymerase in the absence or presence of ammonium sulfate, according to the procedures ofWidnell and Tata." 6, 14 Impure nuclear fractions were prepared by omitting the step of low-speedcentrifugation in 0.32 M sucrose. Both polymerase reactions were dependent upon the presence ofall four nucleoside triphosphates and were inhibited by DNase or actinomycin D.

Preparation of nuclei for autoradiography: Isolated nuclei were incubated in a small disposablepipette, the tip of which had been fused previously by heating. Incubation of nuclei in the pres-ence of MNg2+ and the absence of ammonium sulfate was terminated by rapid cooling to 0-20C.The nuclear suspension was layered beneath with 3 vol 3% glutaraldehyde acrolin in sodium cacody-late (0.24 Al, pH 7.4). Nuclei were pelleted at 0-20C by centrifugation at 200 X g for 2 min, andcollected by breaking the tip of the pipette. The nuclear pellet was then transferred to 1%osmium tetraoxide for postfixation. The preparation was passed through an alcohol series (5min at each step) for dehydration, and embedded in Epon Araldite.

Nuclei to be incubated in the presence of M\n2+ and ammonium sulfate were first pelleted in around-bottom test tube. The sucrose was carefully pipetted off, and the incubation mediumwithout radioactive triphosphate was added. After 15 min of preincubation, the incubationmedium was replaced with the medium containing radioactive triphosphate. At various timesafter preincubation, polymerase reaction in the nuclei was terminated by chilling and the addition

1371

Dow

nloa

ded

by g

uest

on

Mar

ch 8

, 202

0

1372 BIOCHEMISTRY: MAUL AND HAMILTON PROC. N. A. S.

of 1 ml of the glutaraldehyde fixative described above. By this procedure the nuclei remainedattached to the bottom of the test tube, and the incubation medium could be successively dilutedand removed by serial additions of the fixative. Preliminary studies showed that this method ofincubation and fixation of nuclei reduced the damage and dispersion of the nuclear componentsthat resulted from more vigorous incubation techniques. Postfixation, dehydration, and embed-ding of the nuclear material was as described above. Sections were collected on Formvar-coatedcopper grids, and a thin carbon film was added. For high-resolution autoradiography, Ilford L4photographic emulsion was placed on the sections by the loop method of Caro and van Tubergen.'5Exposure time was 2 months for sections of nuclei incubated at low ionic strength, and 4 or 5months for those of nuclei incubated in the presence of ammonium sulfate. After development inMicrodol X for 3 min at 20'C or in Dectol 19 for 2 min at 20'C, fixing, and washing, the sectionswere poststained for 30 min with a 0.5% aqueous solution of uranyl acetate.

Electron micrographs were made by use of Siemens Elmiskope I., without removing the gela-tin of the emulsion. Initial magnification was 3000-5000. For autoradiography at the lightmicroscope level, 0.5 MA sections were cut, placed onto clean slides that were dipped in Ilford L4photographic emulsion (diluted 1:1 with distilled water). After 4 days, the sections were de-veloped for 5 min in Dectol 19, fixed, and washed. Observations were made by use of a Zeissphase-contrast microscope. Counts of grains were made for 200 labeled nuclei of preparations oftwo different slides. A nucleus was considered to be labeled if it showed at least two grains andthe nucleolus was visible.

Results.-Figure 1 shows the time course of activity at 300 or 370C for theMg2+-activated RNA polymerase in purified or impure nuclear fractions. Theactivity of the RNA polymerase in purified nuclei assayed at low ionic concentrationwas more than twice that of the polymerase in cytoplasmically contaminated nuclei

12__ ,IIzcz/a'tEE

o Q-QincubationC 30C

X -** purified fraction TIEAFTER PREINCUBATtON

5 10 15 30 45 mi. 60TIME OF INCUBATION FIG. 2.-Time course for the Mn2+-(NH4)2-504-activated RNA polymerase reaction in

(purified) liver nuclei incubated at 30° orFIG. 1.-Time course for the Mg3+.activated 370C. Incubation mixtures contained in a

RNApolymerase reaction in purified or impure final volume of 0.5 ml: 0.1 ml nuclear sus-liver nuclear fractions incubated at 300 or pension containing about 0.1 mg DNA, 50370C. Incubation mixtures contained in a etmo-esTris-HCl buffer (pH 7.5), 2.0t mo3esfinal volume of 0.5 ml: 0.1 ml nuclear sus- MnCl2, 0.05 ml saturated (NH4)204 (pH 7.5),pension containing about 0.1 mg DNA, 50 0.3 ,u mole ATP, GTP, and CTP. After pre-,umoles Tris-HCl buffer (pH 8.5), 2.5 moles incubation for 15 m3, 40 c UTP-H3 wereMgCl2, 0.3 pmole ATP, GTP, CTP, and 4 added and the incubation continued untilTcUTP-H3 (1.6 c mmole). Activity was termination of the reaction at the times mdi-

quantitated as cpm UTP-H3 incorporated into cated. Activity was quantitated as cpmRNA per mg DNA. UTP-H3 incorporated into RNA per mg DNA.

Dow

nloa

ded

by g

uest

on

Mar

ch 8

, 202

0

VOL. 57, 1967 BIOCHEMISTRY: MAUL AND HAMILTON 1373

similarly assayed. As expected, the polymerase activity increased less rapidly at300 rather than at 370C. At the lower incubation temperature, however, a highlevel of incorporation was reached from 15 minutes to 1 hour. This trend was ob-served both for impure and pure nuclear fractions. The decreased activity of the:\IgI+-activated polymerase after 15 minutes of incubation at 370 suggests pro-nounced degradation of newly formed RNA.4 This reduced activity is presumablya result of RNase activity.

Figure 2 shows the time course of activity at 300 and 370C of the Mn2+-(NH4)2-S04-activated RNA polymerase in purified nuclei. The time course is linear asdescribed by Widnell and Tata,5 6 and again there is evidence that at 30° thepolymerase activity rises less rapidly, but is maintained longer, than at 370C.

A ' 9 B

so~~ ~ ~ ~ ~~~peecofe 5m Mg an the absec of

As

Figure3 shows theappeaanceunde FIG. 3.-Elect ron micrographs showing thenuleus befe i batna effect on nuclei of incubation at 370C in the-x As.J~t]_

presence of 5mM Mg2+and the absence ofbationunderthese conditions was eitamm oniumsulfa te.(A) Nucleus before in-

rison to te nucr c n pcubation. X6,400. (B) Nucleus incubated+ >s-if2* < -4 ai 15 min. X 11,200. (C) Nucleus incubated 1

twAarbad lwtN~s s J*|xo*- l hr. X4,800.

ON.

Figure 3 shows the appearance under the electron microscope (EMI) of the livernucleus before incubation and at 1o and 60 minutes of incubation at 37°C in thepresence of 5 m~l\ \lg2+ and the absence of ammonium sulfate. The effect of incu-bation under these conditions was either to disintegrate or to condense the chromatinin comparison to the nuclear condition prior to incubation.

Dow

nloa

ded

by g

uest

on

Mar

ch 8

, 202

0

1374 BIOCHEMISTRY: MAUL AND HAMILTON PROC. N. A. S.

Figure 4 shows by EKM autoradiography that the nucleolus is the primary siteof activity for the 1\JIg2+-activated RNA polymerase reaction. By 2.5 minutes

A W+ s ^ B = 0 .'

s4 v - w >a} i.s1

4, iv >|_ j r .-* >,,'

Ot''; '' 4 qt'# X ', * i4 i #' 4 ~

g:<~~ ~ ~ ~ ~ ~ ~ AA''A§§ ) * F

At <>7~~~~

HiC:49>t~~~~~~~~~.y.4...thr.X4,800. 9

*r~~~~~~~~~V

S.~~~~ $

4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~f

%IL~~~

AAt

FI4EMauordogaps hoin heloatonofM2 -atiatd N plyerseiliernuleicuatdat37. A)Nule icbaedfo 25 ii. (8,00 () uceiinu

hr.X 4,800 ~~~.

Dow

nloa

ded

by g

uest

on

Mar

ch 8

, 202

0

VOL. 57, 1967 BIOCHEMISTRY: MAUL AND HAM1ILTON 1375

there was pronounced incorporation in the nucleolar region. During incubationperiods up to 1 hour, only very little incorporation occurred in the extranucleolarchromatin (cf. Figs. 4 and 6).The electron micrographs of Figure 5 show the time course for effects of incubation

in the presence of 4 mM i\1n2+ and 0.4 ill ammonium sulfate on liver nuclei. Underthese conditions of incubation, the structure of the nucleus was altered as expected.The positions of the nuclear components, however, were approximately the sameas those of the unincubated nucleus (Fig. 3A). Preliminary results indicated thatthe procedures of incubation, fixation, and postfixation as described in Materialsand Methods minimized the dislocation of the nuclear components. A study by EATof serial sections of nuclei exposed one minute to _Mn2+ and the salt revealed thatthe nucleolus contained a core with granular material separated as a corona (e.g.,see Fig. 5A). The condensed chromatin attached to the nuclear envelope did notdisperse completely even by one hour of exposure to the salt. The electron opaqueareas of the nuclei shown in Figure 5 are thought to be a result of the condensedchromatin or chromatin associated with the nucleolus. The small round structureof Figure 5D is thought to represent the inner core of the nucleolus (cf. Fig. 5C).

Figure 6 demonstrates that incorporation of uridine triphosphate-H3 (UTP-H3)into RNA by polymerase activated by i\1n2+ and ammonium sulfate occurs pri-marily in the chromatin region, and not in the nucleolus. Only very little labelingcould be detected in the region of the nucleolus or of its fragments. By far themajority of the grains were seen over finely dispersed chromatin. In order to havean appreciable number of grains observable, an exposure time of four or five monthswas required. Nearly all nuclei examined showed grains, ranging from 2 to 15 pernucleus. Background was most negligible in the section areas not containing nuclei.

Finally, an attempt was made to compare biochemical and autoradiographicevidence concerning RNA synthesis underwritten by M\Ig2+-activated RNA poly-merase in the nucleolar and extranucleolar regions of the nucleus. Figure 7 com-pares the distribution of nucleolar and extranucleolar grains resulting from 2.5 to15 minutes of incubation of nuclei at low ionic concentration. The increase in totalnuclear grains observed in the sections was in good agreement with the biochemicaldata (cf. Figs. 1 and 7). After 5 minutes of incubation, the majority of grain-indicated nucleolar incorporation was completed, whereas the extranucleolar incor-poration increased slowly from 5 to 15 minutes. It may be important that about 5per cent of the extranucleolar grains were observed over the nuclear membrane.

Discussion. Huang, Bonner, and Mklurray7 and others have demonstrated thatan ionic concentration of 0.4M solubilizes histones from DNA. Chambon, Ramuz,and Doly8 observed a stimulation by ammonium sulfate of RNA synthesis by aDNA-RNA polymerase-histone complex. From one point of view, these observa-tions suggest that the action of ammonium sulfate in stimulating RNA synthesisoccurs by removal of histone from DNA. This would result in greater DNAtemplate activity or gene activation. It is, however, still uncertain how removalof histones from DNA would alter the pH optimum and ionic activation of theRNA polymerase.6 On the other hand, the salt at high ionic concentration mightaffect the activity ofRNA polymerase in the chromatin region of the nucleus. Thisalternative explanation for the action of ammonium sulfate faces the question ofwhy RNA polymerase in the nucleolus is not similarly affected. Different RNApolymerases in terms of activation requirements as well as intranuclear positions

Dow

nloa

ded

by g

uest

on

Mar

ch 8

, 202

0

1376 BIOCHEMISTRY: MAUL AND HAMILTON PRoc. N. A. S.

W'!,q ":UPS

z Ame x-:~~~~~~~~~~-.'k ~~~~~~~~~~~~~~~~~~~~~~~~A

R~ ~ ~ SraA~~~~~c7_~~~~~~~~~~~~~~~~~~~~~~~~~.

4 */J

`OM~

.4,

V PhV .

P~~~

V'

N~~~~~~~~~~~~~~~~~~~~

N~~~~~~~~~~~~~pitoRtt

~~~94 A~~~~~~~~~~

fragmented~~~~~~~~~~~~~~~~~~AnulolsX4,00

Dow

nloa

ded

by g

uest

on

Mar

ch 8

, 202

0

VOL. 57, 1967 BIOCHEMISTRY: MAUL AND HAMILTON 1377

' ~ ~ . h : ...,41,¢..,

*_. 5

41. 4.~~~~~~~~~~W4

s ~~~~~~**4 5 . - *0s ..+'

:. ~, S'4

"D ,t * s

A~~~~~~~~~~~~~~~~~~~j-? A

*- - 4.... ,

S. . ,S

A'W4, V f <~~~~4 '~~4 '1 Ps;

4 . 1;,b , '.. w*

k r~~Ar

" .:.

C' . ni.y

of a nucleus inuae in th prsec of Mn +*n th sal bu in th abec;fraiatv

triphosphate. 2<4,480.-n41

FIG~~~~~~~~u^,0'..-K.,oado h shwin th loato 'or '\n+(H)S -_ctivate RN"oy

merase in liver nuclei incubated for 1 hr at 37°C. (A) Autoradiograph exposed for 4 months.X6,080. (B) Autoradiograph exposed for 5 months. X5,440. (C) Autoradiograph exposed for4 months but developed with Phenidonell for 1 min at 24°C. X6,400. (D) Electron micrographof a nucleus incubated in the presence of Mnl+ and the salt, but in the absence of radioactivet riphosphate. X 4,480.

Dow

nloa

ded

by g

uest

on

Mar

ch 8

, 202

0

1378 BIOCHEMISTRY: MAUL AND HA.MILTON Paoc. N. A. S.

t100 _ |> :

> total80-nuclear : FIG. 7.-A comparison of the dis-

.o grains 6 tribution of nucleolar and extranucle-0 // _ <,, olar grains in relation to total nuclear

nucleolar * grains during the 15-min course of:360 grains > the M\rg2+-activated RNA polymerase

o i 4 reaction. Acid-insoluble radioactiv-: / // i,, ity is expressed as percentage of the

440 /E activity manifest at 15 min of incuba-/tion (Fig. 1). Two hundred nuclei

o 2// were counted for each period of incu-ID - - 2 bation designated. At 15 mim the

V20' extran cleolar average number of grains per nuclettswas 7.94.

0 2.5 5.0 10.0 min. 15.0TIME OF INCUBATION

could be one answer to the question. Multiple effects of the salt would not be un-expected. However, statements such as these must remain speculatory until preciseinvestigations can be made using isolated purified enzyme preparations of mam-malian RNA polymerase(s).Sunimary.-The intranuclear localization of two DNA-dependent RNA polym-

erase reactions was investigated by high-resolution autoradiography. TheM\Ig2+-activated RNA polymerase reaction that synthesizes ribosomal RNA occursprimarily in the nucleolus. The Cn2n+-(NH4)2SO4-activated RNA polymerasereaction that synthesizes a more DNA-like RNA occurs primarily on the chromo-somes in the extranucleolar region.

Note added in proof: Since the communication of our paper, similar results have been reportedby A. 0. Pogo, V. C. Littau, V. G. Allfrey, and A. E. MIirsky (these PROCEEDINGS, 57, 743 (1967)).

* This research was supported by a U.S. Public Health Service research grant (HD-00726-05to T. H. H.), and a Rosalie B. Hite predoctoral fellowship (to G. G. -M.). We thank BeverlyJohnson and Donna Gerald for excellent technical assistance. We are indebted to J. J. Bieselefor his interest and assistance in various ways.

1 Weiss, S. B., these PROCEEDINGS, 46, 1020 (1960).2 Huang, R. C., N. -Maheshwari, and J. Bonner, Biochemi. Biophys. Res. Commun., 3, 689

(1960).3Goldberg, J. H., Biochin. Biophys. Acta, 51, 201 (1961).4 Biswas, B. B., and I. Abrams, Biochim. Riophys. Acta, 55, 827 (1962).5 Widnell, C. C., and J. E. Tata, Biochim. Biophys. Ada, 87, 531 (1964).6 Ibid., 129, 478 (1966).7 Huang, R. C., J. Bonner, and K. M\1urray, J. Mol. Biol., 8, 54 (1964).8 Chambon, P., AI. Ramuz, and J. Doly, Biochem. Biophys. Res. Commun., 21, 156 (1965).9 Perry, R. P., these PROCEEDINGS, 48, 2179 (1962).

10 MIcConkey, E. MI., and J. W. Hopkins, these PROCEEDINGS, 52, 931 (1964).11 Liau, M. C., L. S. Hnilica, and R. B. Hurlbert, these PROCEEDINGS, 53, 626 (1965).12 Busch, H., P. Byvoet, and K. Smetana, Cancer Res., 23, 313 (1963).13 Ro, T. S., AM. Murmatsti, and H. Busch, Biochem. Biophys. Res. Commun., 14, 149 (1964).14 Widnell, C. C., and J. R. Tata, Biochem. J., 92, 313 (1964).15 Caro, L. G., and R. P. van Tubergen, J. Cell. Biol., 15, 173 (1962).16 Lettre, H., and N. Paweletz, Naturwissenschaften, 53, 268 (1966).

Dow

nloa

ded

by g

uest

on

Mar

ch 8

, 202

0