Effects of ionic strength on the RNA polymerase activities of isolated nuclei and nucleoli of rat...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 142, 489-%@ (1971)

Effects of Ionic Strength on the RNA Polymerase Activities of

Isolated Nuclei and Nucleoli of Rat liver

JOHN D. JOHNSON,2n3 BARBARA A. JANT, SEYMOUR KAUFMAN, AND

LOUIS SOKOLOFF

Laboratory of Cerebral Metabolism and Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 80014

Received September 28, 1970; accepted November 23, 1970

With appropriate divalent cation concentrations the RNA polymerase activity of intact nuclei isolated from rat liver exhibits a biphasic stimulatory response to increasing ionic strength. Base ratios and susceptibility to inhibition by low concentrations of actinomycin D indicate that the initial peak of stimulation at 40 mM (NH&S04 represents enhancement of pre-rRNA synthesis whereas the second phase of stimulation at lo-fold higher ionic strength reflects increased synthesis of more DNA-like RNA species. Evidence has been obtained that the initial peak reflects an effect on nucleolar RNA polymerase activity. Isolated nucleoli exhibit only a monophasic stimulation of pre-rRNA synthesis in response to increasing ionic strength; the peak occurs at 60 DIM (NH&SOa and appears to correspond to the initial peak observed at 40 mM (NH&SO 1 with intact nuclei. These findings suggest not only that intact nuclei from rat liver incubated in vitro synthesize a mixture of pre-rRNA and DNA-like RNA, but also that both pre-rRNA and DNA-like RNA synthesis can be stimulated separately by different levels of ionic strength.

Numerous physiological influences are known to operate on the activity of nuclear RNA polymerase [nucleoside triphosphate : RNA nucleotidyl transferase [DNA dependent]; EC 2.7.7.61 in mammalian cells. These include hormones (2), nutritional status (3), developmental age (4-6), and specific organ responses such as liver re- generation and cardiac and skeletal muscle hypertrophy (7-10). Cytoplasm from various rapidly growing tissues has recently been shown to enhance the in vitro RNA polymerase activity of nuclei derived from slowly growing cells (11). The mechanisms which mediate these influences are still unknown. A number of studies have shown that ionic

1 A preliminary account of portions of this work has been reported (1).

2 Present address : Department of Pediatrics, Stanford University School of Medicine, Palo Alto, Calif.

3 Staff Associate of the NICHD during the course of these studies.

strength exerts a potent influence on RNA polymerase activity in vitro (12-17). Very high ionic strength has been found to enhance DNA-like RNA4 synthesis (12-15), but no effects of ionic strength on the synthesis of pre-rRNA4 have been reported.

In the course of studies on cytoplasmic influences on nuclear RNA synthesis, it was found that under certain conditions ionic strength, at levels within the physiological range, may also strongly influence the rate of pre-rRNA synthesis. It was noted that with intact nuclei in vitro progressively increasing levels of ionic strength produced two separate peaks of stimulation of RNA polymerase activity, one at relatively low

4 The abbreviations used are: pre-rRNA, preribosomal RNA (GMP + CMP content close to 70%); DNA-like RNA, RNA richer in AMP + UMP content compared to that of pre-rRNA and more complementary to the base composition of DNA; AU/GC, ratio of AMP + UMP:GMP + CMP.

489

490 JOHNSON ET AL.

ionic strength [e.g., 40 mM (NHI)&304] and the other at IO-fold higher ionic strength (1). Preliminary studies (1) indicated that the stimulation by low ionic strength was exerted on the synthesis of RNA with a base composition similar to that of pre-rRNA, whereas, in agreement with previous reports (12-15), high ionic strength stimulated the synthesis of more DNA-like RNA. The present report examines these effects with intact nuclei in greater detail and describes the results of similar experiments with isolated nucleoli. The studies with nucleoli demonstrate only a monophasic stimulation of nucleolar RNA polymerase activity by (NHJ$Od which appears to be equivalent to the stimulation observed in intact nuclei with low concentrations of (NH,), SO,. This stimulation does not alter the base composition or the properties of the newly synthesized RNA from those of pre-RNA. The results of the present studies establish that nucleolar RNA synthesis can be stimulat#ed by ionic strength and that the stimulation of nuclear RNA polymerase activity by low concentrations of monovalent cations represents enhancement of nucleolar synthesis of pre-rRNA. The stimulation of DNA-like RNA synthesis by high concentrations of (NHJzS04 does not occur in nucleoli and appears, therefore, to be an effect on nucleoplasmic RNA synthesis.

MATERIALS AND METHODS

Chemicals

Uniformly labeled (I%) ribonucleoside triphosphates and erotic acid-6-l% were purchased from Schwarz BioResearch. Unlabeled ribonucleoside triphosphates were obtained from P-L Biochem- icals. Actinomycin D was provided by Merck, Sharp and Dohme. Yeast RNA was obtained from Sigma Chemical Co., salmon sperm and calf thymus DNA from Calbiochem, and crystalline bovine serum albumin from Pentex.

Procedures

Preparation of nuclei. Normal Osborne-Mendel male rat,s, weighing 175-200 g and fed ad l&turn on Purina Laboratory Chow, were used in all experiments. Nuclei were isolated by the method of Widnell and Tata (18). Liver was homogenized in 0.32 M sucrose-3 mu MgClz solution, filtered through 70.mesh nylon fabric and centrifuged at 750g for 10 min. The crude nuclear pellets were

suspended in 2.4 M sucrose-l mu MgClz and centri- suged at 50,ooOg for 60 min in a Spinco Model L preparative ultracentrifuge. The purified nuclear pellets were then further washed twice by resuspension and centrifugation (7509) in ice-cold 0.25 M sucrose-l mM MgClz solution, and finally resuspended in the same medium at a concentration of 1.0-4.0 mg of nuclear DNA/ml.

Preparation of nucleoli. For isolation of nucleoli, a modification of the procedure of Mura- matsu, Smetana, and Busch (19) was employed. After the nuclei were centrifuged through 2.4 M sucrose containing 1 mM MgC12, they were resuspended in 1.5 ml of 0.25 M sucrose solution/l g of original wet liver weight or a volume sufficient to yield a final concentration of approximately 0.5 mg of nuclear DNA/ml. Aliquots of 15 ml were sonified with a Branson Model LS-75 sonifier operated at 5.5 A for lo-set intervals. The samples were chilled during sonication by immersion in a Dry Ice-ethanol bath kept at -10 to -15’. The degree of lysis of the nuclei was monitored by phase contrast or light microscopy; lysis of greater than 99% usually required 5&60 set under these conditions. Each aliquot of the nuclear sonicate was centrifuged through a 15-ml layer of 0.88 M sucrose at 20009 for 15 min (Sorvall RC-2B centrifuge; HB-4 swinging bucket rotor). The nucleolar pellets were washed by resuspension in 0.25 M sucrose and centrifuged again through a layer of 0.88 M sucrose. The final washed pellets were suspended in 0.05-0.1 ml of 0.25 M sucrose solution/l g of original wet liver weight.

Electron microscopic examination demonstrated that the isolated nucleoli maintained their structural integrity and were free from significant contamination. Table I compares the chemical composition of the nuclear and nucleolar fractions employed in these studies; the charac- teristics of both were similar to those previously reported in the literature (12, 16, 18-21).

RNA polymerase assay. The standard assay contained the following components in a final volume of 0.25 ml: 0.1 M Tris-HCl, pH 8.1 at 23’; 10 mM 2-mercaptoethanol; 0.4 mM ATP, UTP, GTP, and CTP with one of the triphosphates labeled with 14C at a specific activity of 2.0 pCi/ pmole; varying concentrations of MgCI,, MnCl*,

and (NHI)~SO~ as indicated in the figures and

tables; and nuclei containing 100-200 pg DNA or nucleoli containing 5-15 pg DNA. Incubations

were carried out in a shaking water bath at 30” for 10 min. The reaction was stopped by the addition

of 5 ml of cold 5yo (w/v) trichloroacetic acid-O.02 M Na4P207. In studies with whole nuclei, the acid-

precipitable residues were collected by centrifugation and washed three times by resuspension

491 RNA SYNTHESIS BY RAT LIVER NUCLEI AND NUCLEOLI

TABLE I CHEMICAL CHARACTERIZATION OF RAT LIVER NUCLEI AND NUCLEOLI"

Ratio Nuclei Nucleoli

DNA/RNA 6.35 & 0.69 (13) 0.70 f 0.06 (8) Protein/RNA 20.8 f 1.93 (5) 7.0 f 0.46 (6) Protein/DNA 3.06 f 0.33 (5) 10.3 + 1.3 (6)

a The ratios (weight per weight) given are the means f. standard errors for the number of preparations shown in parentheses. Chemical determinations were performed as described in Procedures.

in 5 ml of 5% trichloroacetic acid-O.02 M NadP*OT and recentrifugation. The washed pellets were suspended in 1.0 ml of 0.2 M Tris-HCl, pH 8.0 at 23”, dissolved by the addition of 20 ~1 of 9 N NaOH and immediately reprecipitated by the addition of 5 ml of cold 10% (w/v) trichloroacetic acid. The reprecipitated material was collected by centrifugation and dissolved in 0.5 ml of Nuclear- Chicago solubilizer (NCS). A toluene-phosphor solution was added, and the radioactivity was determined in a Packard Tri-Carb scintillation spectrometer with approximately 75y0 efficiency. In the studies with the isolated nucleoli, the reac- tions were terminated by the addition of 5% trichloroacetic acid-0.02 M NadP207, and the acid- precipitable material was collected by centrifugation and then transferred to Whatman GF/C glass fiber filters. The filters were washed succes- sively with 15 ml of cold 5% trichloroacetic acid- 0.02 M Na4Pz07, 5 ml of 95% ethanol, 5 ml of di- ethylether and then placed in counting vials. Ten milliliters of the toluene-phosphor solution were added, and the radioactivity was measured in the liquid scintillation counter with 65-70% efficiency under these counting conditions. The results are expressed as mpmoles of (1°C) nucleotide incorporated in 10 min/mg of DNA. Under the above conditions, the specific activity of the precursor (1°C) nucleotide was 3500-3800 cpm/ mpmole .

The RNA polymerase activity in both the nuclear and nucleolar preparations ws~9 dependent on all four nucleoside t,riphosphates for maximal activity and was inhibited by pancreatic RNase, DNase, Na4Pz07, and actinomycin D. More than 99% of the radioactive product was rendered acid soluble by treatment with 0.3 N KOH for 18 hr at 37”. Both initial velocity and total (14C) nucleotide incorporation were greater at 30” than at 37”, a finding similar to that of Maul and Hamil- ton (22) and of Meisler and Tropp (16). The reaction velocity was linear for 5 min under the conditions employed, and all the differences in activity reported here represent changes in initial velocity as well as the extent of the reaction. The concentrations of the nucleotides (0.4 mM) were

at lea& 10 times the amount required to achieve half-maximal velocity.

Base composition analysis. Reaction mixtures and incubation conditions similar to those of the standard assay were employed, except that all four nucleoside triphosphates were labeled with 14C at a specific activity of 5-20 pCi/pmole. In any one experiment, the specific activities of the four nucleotides were adjusted to be equal. The acid-precipitable material was washed four times with 5% trichloroacetic acid-O.02 M Na4P207, dissolved with NaOH and reprecipitated with 10% trichloroacetic acid in the presence of 0.5-1.0 mg of yeast RNA added as carrier. The precipitates were then usually dissolved in 0.5 ml of 0.3 N KOH and incubated at 37” for 18 hr to hydro- lyze the RNA to 2’(3’)-mononucleotides; in some experiments the precipitates were further extracted twice with 10% (w/v) NaC1-0.05 M Tris- HCl (pH 8.0 at 23”) at 100” for 30 min, and the RNA reprecipitated with 2 vol of 95% ethanol before alkaline hydrolysis. The final results obtained with the two procedures were almost identical. The base hydrolysate was acidified with 50 pl of 6 N perchloric acid and centrifuged. The supernatant solution was neutralized with 2 N

KOH, acidified to 0.05 N HCl, and the mononucleotides were then separated according to the procedure of Katz and Comb (23). One-milliliter aliquots of each of the column fractions were added to 10 ml of Omniflour-toluene (4 g Omni- fluor/l. of toluene) phosphor solution containing 2 ml of Beckman Biosolv BBS-3, and the radioactivity was measured in the liquid scintillation spectrometer with 75-80y0 counting efficiency.

The specific activity and radiochemical purity of the nucleoside triphosphate substrates were determined by Dowex l-Cl column chromatography (24) or thin layer chromatography on DEAE-cellulose plates (25).

RNase a&z&y. Animals were injected intra- peritoneally with erotic acid-6-l% (36.5 mCi/ mmole, 15 &i/100 g of body wt) 20 min prior to sacrifice. Nuclei and nucleoli were then isolated as described above and incubated in reaction mixtures identical to those used for the RNA poly-

492 JOHNSON ET AL.

I I I I I I I I I , I I I I I

z A. No added solt B. 40mM (NH412S04 C. 400 mM (NH4)2.504

g 4.0- NUCLEI

F ‘; 3.0- o----o Mn2+ .- E

e 2.0-

- M$+

- o , +yL , , I I I I I I I I I I

F

I I I I I

2 2 20-

NUCLEOLI

E --79

I I

0 2 4 6 8 IO 0 2 4 6 8 IO

CATION CONCENTRATION ( mM 1

FIG. 1. Effects of divalent cation concentrations on RNA polymerase activity at various ionic strengths. RNA polymerase activity was determined as described in Procedures with (VZ) UTP as the radioactive precursor. Either 120 pg of nuclear DNA or 5 pg of nucleolar DNA were present in each incubation mixture. The concentrations of Mg2+ (a-- 0) or Mn2+ (O--O) varied as indicated.

merase assay, except that none of the nucleoside triphosphates was radioactive. After incubation for 10 min at 30”, the reaction was stopped by the addition of 1.0 ml of cold 5% trichloroacetic acid- 0.02 M NadP207. After standing for 20 min, the acidified reaction mixtures were filtered through Whatman GF/C glass fiber filters which were then washed twice with 0.5 ml of cold 50/o trichloroacetic acid-O.02 M NadP207. The filtrates were collected and mixed with 15 ml of Omnifluor- toluene (4 g/l.)-Beckman Biosolv BBS-3 (5:l v/v) phosphor solution. Radioactivity was measured in the liquid sintillation counter. Omission of the nucleoside triphosphates, which were routinely added to simulate exactly the conditions of the polymerase assay, had no effect on the assay of RNase activity. Release of acid-soluble radioactivity was linear with time for at least 20 min and was directly proportional to the amount of %-labeled nuclei or nucleoli added to the incubation mixture, at least, in the range employed.

6 Qualitatively similar results for nucleolar RNase act,ivity have been obtained with exoge- nous yeast ‘4C-labeled ribosomal RNA as the substrate. However, the quantity of acid-soluble radioact,ivity released with this substrate is 75-100

More than 99% of the radioactivity contained in the labeled nuclear and nucleolar preparations was acid precipitable; 98yo of the radioactivity was rendered acid soluble after base hydrolysis (0.3 N KOH, 18 hr at 37”). In the nucleoli, 8% of the radioactivity was found to be in CMP, and 92% in UMP; the distribution was 5% in CMP and 95% in UMP in the nuclei.

Miscellaneous procedures. RNA was measured in samples of the nuclear and nucleolar suspen- sions by the method of Fleck and Begg (26). The washed residues remaining after base hydrolysis and acidification were extracted twice with 0.5 N perchloric acid at 70” for 15 min, and DNA was determined by the Burton modification of the diphenylamine procedure (27) with salmon sperm or calf thymus DNA used as standards (9.0% phosphorus). Protein was determined according to Lowry et al. (28) with crystalline bovine serum albumin as the standard.

times the amount of nucleotide incorporation into RNA obtained with the RNA polymerase assay for the same number of nucleoli [also see (IS)]. We, therefore, feel that the degradation of newly synthesized endogenous RNA is a more valid assay for the RNase activity for the purposes of the present studies.

RNA SYNTHESIS BY RAT LIVER NUCLEI AND NUCLEOLI 493

RESULTS

Influence of divalent cations on nuclear RNA polymerase activity. RNA polymerase activity in intact nuclei was only partially dependent on added divalent cations; 25- 50% of the maximal activity was observed in the absence of any added Mgz+ or Mn2+, and the degree of activation achieved on their addition was influenced by the ionic strength of the medium (Fig. 1). At high ionic strength both Mg”+ and Mn2+ were almost equally effective as activating divalent cations; the maximal activities achieved with both were essentially the same although reached at lower concentrations of Mn2+ than Mg2+ (Fig. 1C). At low ionic strength, however, Mn2+ was clearly the preferred species (Fig. 1A and B) ; the maximal activities were always greater and the optimal concentrations smaller with Mn2+ than with Mg2+.

Another difference between Mg2+ and Mn2+ was in the influence of ionic strength on the shape of the activity-concentration curves. Mg2+ exhibited a broad peak of activity at all ionic strengths; on the other hand, the sharp peak observed with Mn2+ at low ionic strengt,h broadened at high

ionic strength and assumed a configuration like that obtained with Mg2+. This effect of ionic strength on the activity-concentration relationships for Mn2+ has been observed by others in intact nuclei and also in the aggregate enzyme (13,15,17).

InjEuence of ionic strength on nuclear RNA polymerase activity. The RNA polymerase activity of whole nuclei exhibits a biphasic stimulation in response to increasing ionic strength (Fig. 2) (1). A relatively narrow peak of stimulation is observed at about 40 mM (NH4)2S04, and a second broader peak occurs at higher concentrations, increasing to a maximum between 300-400 mM. These effects are related to ionic strength rather than species or concentration of the salt; KCl, for example, produces qualitatively similar patterns but at the higher molar concentrations required to yield the same ionic strengths. The stimulations by ionic strength require the presence of appropriate concentrations of divalent cations, but similar results are obtained with either Mg2+ or Mn2+, added singly or in combination (1). Excessive concentrations of Mn2+, which inhibit RNA polymerase activity at low ionic strength, also eliminate the st8imulation

Nuclei - --- _--- -2

P- 3 / 0

/ ci

I I I I 0 80 160 240 320 400

(NH4 ) +04 CONCENTRATION ( mM)

FIG. 2. Effects of ionic strength on RNA polymerase activity in nuclei and nucleoli. The incorporation of (1%) UTP into acid-insoluble material was determined as described in Procedures. Either 115 rg of nuclear DNA or 5 pg of nucleolar DNA were present in each reaction mixture. A-A, whole nuclei, 2.7 rnM Mg2f plus 0.6 rnM Mn *; a-0, nucleoli, 0.6 mM Mn2+; O--O, nucleoli, 2.5 rnM Mg2+.

494 JOHNSON ET AL.

B. NUCLEOLI

FIG. 3. Effects of Mnz+ concentration on the response of nuclei and nucleoli to increasing ionic strength. Nuclei (115 pg of DNA per incubation tube) or nucleoli (6 rg DNA per incubation tube) were assayed for RNA polymeraae activity tt9 described in Procedures in the presence of varying concentrations of MnClz and (NH&SOI with (14C) UTP m the radioactive precursor. +----a, nuclei, 0.6 rnM Mn2+; O---O, nuclei, 1.2 mu Mn 2+; A-A, nuclei, 2.4 mM Mn2*; A-A, nuclei, 3.0 mu Mn*+; O-e, nucleoli, 0.6 mM Mn*; O- 0, nucleoli, 1.5 mM Mn*+; A----A, nucleoli, 3.0 mM Mn”.

produced by 40 mu (NH4)804 (Fig. 3A) ; excessive Mg”+ concentrations have similar effects.

Efects of ionic strength on the base composition of newly-synthesized RNA in nuclei. Base composition analysis indicated that the stimulation of RNA synthesis by low ionic strength represented predominantly pre- rRNA. The AU/GC ratios, especially in the presence of Mn2+, were slightly higher than might be expected for pure pre-rRNA (29-32), indicating some contamination by other types of RNA, but the stimulation by low ionic strength was associated with a tendency to shift the ratio toward one more characteristic of pre-rRNA.

On the other hand, the RNA newly synthesized in the presence of 400 mM (NH&SO4 w&s enriched in AMP and UMP contents, indicating that it was more DNA- like (Table II).

Efects of actinomycin D on nuclear RNA polymerase activity. Additional evidence re- garding the types of RNA involved in the stimulations by the two levels of ionic strength was obtained from studies with actinomycin D. At low concentrations this agent preferentially inhibits the synthesis of pre-rRNA (13, 33-35). When added to our nuclear RNA polymerase assay system, such

low concentrations of actinomycin D inhibited RNA synthesis most markedly at the peak of the stimulation produced by low levels of ionic strength (Table III). The effect of the agent was, therefore, to suppress more or less preferentially the stimulation of RNA synthesis by 40 mM (NH&S04 (Table III), and this suppression was ac- companied by a shift in the base composition of the newly synthesized RNA closer to that of DNA-like RNA (Table II). These results offer additional and independent evidence that (NHd)&Od at low ionic strength stimulates pre-rRNA synthesis.

Nucleolar RNA synthesis. Since pre- rRNA is synthesized in the nucleolus (33), further evidence that pre-rRNA synthesis can be stimulated by changes in ionic strength was sought by studying the RNA polymerase activity of nucleoli isolated from the nuclear preparations. The nucleolar RNA polymerase activity exhibited the same general responses to the divalent cations, Mg2+ and Mn2+, as those observed with intact nuclei, except that the de- pendence on the presence of either one or the other of these cations was absolute (Fig. 1, lower). However, in contrast to the results obtained with nuclei, only a single peak of stimulation was observed with increasing

RNA SYNTHESIS BY RAT LIVER NUCLEI AND NUCLEOLI

TABLE II

BASE COMPOSITION OF RNA SYNTHESIZED BY NUCLEI in wilroa

495

wH4)2so4 Divalent cation giti,“d

Mole % A+U

b.4 A U G C G+C

- 2.7 mM Mgz+ 0 14.5 (16.4) 22.4 (24.4) 34.4 (31.6) 28.7 (27.6) 0.58 (0.69)

40 14.9 (19.4) 21.9 (22.6) 34.1 (30.4) 29.1 (27.6) 0.58 (0.72) 80 13.9 (17.5) 21.7 (23.6) 35.3 (29.9) 29.1 (29.0) 0.55 (0.70)

400 18.3 (19.8) 26.6 (28.2) 30.0 (28.5) 25.1 (23.5) 0.82 (0.92)

1.2 mM Mn2f 0 17.1 (17.3) 25.0 (26.4) 31.5 (31.2) 26.4 (25.1) 0.73 (0.78) 40 16.7 (19.4) 24.9 (27.2) 31.7 (28.0) 26.7 (25.4) 0.71 (0.87) 80 15.4 (17.6) 23.2 (25.2) 32.7 (32.3) 28.7 (24.9) 0.63 (0.75)

400 19.8 (20.2) 27.6 (28.9) 28.0 (26.8) 24.6 (24.1) 0.90 (0.96)

Rat liver pre-rRNA 12.9 19.3 37.2 30.6 0.47 (455) * Rat liver DNA* 28.8 28.7= 21.2 21.3 1.35d

a The assay conditions are described in the section on Procedures. Each reaction mixture contained 200 pg of nuclear DNA and all four (14C) nucleoside triphosphates (5 pCi/pmole, 0.4 mu). Results obtained in the presence of actinomycin D (0.2 pg per reaction mixture, 1 pg/mg of DNA) are shown in parentheses. The values represent the means of duplicates which differed from one another by less than 5yob. The values for the base compositions of total rat liver pre-rRNA and DNA are presented for comparison.

* Values taken from Steele (32). c Value for thymidylic acid content. dA+T/G+C.

TABLE III INHIBITION OF NUCLEAR RNA SYNTHESIS in vitro

BY ACTINOMYCIN D AT VARIOUS

IONIC STRENGTIW

(1%) Nucleotide

(NHdrSO4 incorpixation

Divalent cation concen- (m~moles/lo Actino- min/flask) mycin D

tration effect (4

2.7 mM Mg2+ 0 1.35 0.77 -43 40 2.16 1.07 -51 80 2.26 1.06 -53

400 3.70 3.03 -18

1.2 mM Mn2f 0 1.71 1.32 -23 40 3.21 2.10 -35 80 2.62 1.80 -31

400 4.67 3.82 -18

a The composition of the assay mixtures and the incubation conditions were identical to those described in Table II. Each flask contained 2CO pg of nuclear DNA; when added, the actinomycin D addition was 1 rg/mg of nuclear DNA. Total (W) nucleotide incorporation was determined as described in Procedures. The effects of the actinomycin D on the base composition of the newly synthesized RNA are presented in Table II.

ionic strength (Fig. 2). This peak occurred at approximately 60 mM (NH&S04, corre- sponding relatively closely with the initial peak observed with nuclei; the small differences in optimal (NH4)2S04 concentrations may reflect the fact, that the nucleoli were more completely freed of endogenous ions as a result of the extensive washing in an ion- free medium during their isolation. This effect of ionic strength was essentially the same with optimal concentrations of either Mg2+ or Mn2+.

The base composition of t,he RNA synthesized by nucleoli was similar to that previously reported for pre-rRNA (32) and re- mained unaltered at all levels of ionic strength, even at the peak of stimulation by (NHd)2S04 (Table IV).

Actinomycin D inhibit)ed the nucleolar RNA polymerase activity, and, in contrast t,o the results with intact nuclei, the degrees of inhibition obtained with various concentrations of actinomycin D were the same at all levels of ionic strength (Fig. 4). Further- more, the base composition of the newly synthesized RNA was essentially unaffected by

496 JOHNSON ET AL.

TABLE IV BASE COMPOSITION OF RNA SYNTHESIZED BY NUCLEOLI in vitro IN THE PRESENCE AND

ABSENCE OF ACTINOMYCIN Da

Divalent cation (NmrSO4

concentration (4 AMP

Mole % A+U

UMP GMP CMP G+C

2.5 mM Mge+ 0 10.1 (10.1)

60 10.3 (10.2)

400 10.4 (10.7)

0.6 mM MI?+ 0 10.0 19.8 40.5 (10.3) (20.6) (40.3)

60 10.3 19.9 40.3 (10.5) (20.6) (39.7)

406 10.4 21.1 39.1 (10.6) (21.8) (39.2)

Nucleolar RNA composition 14.4 21.2 36.4 28.0 0.55

(E) 20.0

(20.7) 21.0

(21.6)

39.0 (39.8) 40.0

(39.8) 39.2

(39.3)

30.8 (29.3) 29.7

(29.3)

(E)

29.7 (28.8)

(23)

(iii,

0.43 (0.45) 0.43

(0.45) 0.46

(0.48)

0.42 (0.45) 0.43

(0.45) 0.46

(0.48)

a Base composition of newly synthesized RNA was measured aa described in Procedures. The values represent the means of two experiments with different nucleolar preparations. In both experiments the (1%) nucleoside triphosphate concentrations in the reaction mixtures were 0.4 mM, 8 &i/pmole. In one experiment the reaction mixture contained 30 rg of nucleolar DNA, and tubes with and without 0.1 rg of actinomycin D were paired. In the other experiment the reaction mixture contained 35 pg of nucleolar DNA, and tubes were paired with and without 0.05 rg actinomycin D. Values obtained in the presence of actinomycin D are shown in parentheses. In both experiments total incorporation in the presence of actinomycin D was inhibited 70-75yo under all conditions. The data for “nucleolar RNA” represent the base composition of the total nucleolar RNA, as determined by the procedure of Katz and Comb (23).

I I I I I I I

loo -t l No added salt

I I O 0

I I I I I I I I 2 3 4 5 6

pg ACTlNOMYCtN D/mg NUCLEOLAR DNA

FIG. 4. Inhibition of nucleolar RNA synthesis in vitro by actinomycin D at various ionic strengths. Nucleoli (8.3 pg of DNA per tube) were incubated with 0.6 mM MnClg and varying concentrations of actinomycin D. (1%) UTP was the labeled precursor. Similar results have been obtained with MgClz in place of MnC12.


the actinomycin D inhibition (Table IV). These results demonstrate that nucleolar pre-rRNA synthesis is stimulated by low levels of ionic strength and provide evidence that the stimulation of RNA polymerase activity in intact nuclei by 40 rnM (NH&SO4 represents an increase in nucleolar pre-rRNA synthesis.

RNase activity in nuclei and nucleoli. Rat liver nuclei and nucleoli have been reported to contain RNase activity (15, 16, 36). The incorporation of labeled precursors into RNA may, therefore, represent the net bal- ance between RNA synthesis and degradation, and the influence of ionic strength on (‘“C) nucleotide incorporation into RNA could conceivably be explained by inhibition of RNase activity. The nuclei and nucleoli employed in the present studies were found, in fact, to contain RNase activity which, in agreement with other reports (15, IS), de- clined with increasing ionic strength (Table V). If inhibition of RNase activity were to account for the apparent stimulation of RNA polymerase activity by (NH&SOa, then one might expect lower RNase activities at the salt concentrations at which the stimulations occur. This is clearly not the case with nucleoli; the RNase activity was markedly lower at concentrations of (NH4)$04 beyond the peak of the stimulation of pre-rRNA synthesis. The results were similar with nuclei; RNase activity at 40 mM (NH&S04, the concentration at which the initial stimulation of (‘“C) nucleotide incorporation into RNA is maximum, was greater than that at 80 mM (NH4)zS04, a concentration which lies between the two peaks of stimulation. The increased nucleotide incorporation into RNA at low ionic strength is, therefore, not attributable to inhibition of RNase activity. The effect with 400 mM (NH4)$04 is less clear. At this concentration the inhibition of RNase activity is almost complete and could lead to an apparent stimulation of nucleotide incorporation into RNA. It has been suggested that the entire effect of high ionic strength on RNA synthesis is actually secondary to inhibition of RNase (16, 37). Our data do not contradict this view, but Chambon et al. (15) have found that high concentrations of (NH4)$04 may still stimulate polymerase

TABLE V EFFECTS OF DIVALENT CATIONS AND IONIC

STRENQTH ON RNA DEGRADATION BY NUCLEI AND NUCLEOLI"

Divalent cation wHa)zSO concentra-( Acid soluble Relative tion (mu) cPm activity

Nuclei 2.7 mM Mg2+ 0

40 80

400 1.2 mM Mn*+ 0

40 80

400 3.0 mM Mn2+ 0

938 100 1257 134 977 104

34 4 1085 100 777 72 444 41

51 5 450 -

Nucleoli 2.5 mM Mg2+ 0 325 100

60 219 67 400 7 2

0.6 mM Mn2+ 0 554 100 60 233 42

400 14 3 3.0 mM Mn2+ 0 93 -

a Nuclei (92 pg DNA; 10,600 cpm) or nucleoli (11 pg DNA; 4690 cpm), which had been labeled in ~&IO with (1%) erotic acid, were incubated for 10 min at 30” in the presence of Mg2+ or Mn*+ and varying concentrations of (NH&SO4. The details of the assay for RNase, which is based on the release of acid-soluble radioactivity, are described in Procedures. Zero time values of acid- soluble radioactivity were 48 cpm for nuclei and 9 cpm for nucleoli. Similar results have been obtained in other experiments in which the de- creases in acid-insoluble radioactivity were determined.5

activity under conditions in which RNase activity is absent.

DISCUSSION

Numerous studies have demonstrated that high ionic strength stimulates the DNA-dependent RNA polymerase activity of isolated rat liver nuclei (12-17). The base composition of the RNA synthesized at low ionic strength appears to be that of pre- rRNA, but under the conditions of stimulation by high ionic strength the composition of the newly synthesized RNA becomes more DNA-like in nature (12-15). It is generally believed that the pre-rRNA is nucleolar and

498 JOHNSON ET AL.

the DNA-like RNA nucleoplasmic in origin and that the stimulation by high ionic strength represents an enhancement of RNA synthesis by extranucleolar chromatin (12, 22).

The results of the present studies confirm that high ionic strength leads to increased synthesis of DNA-like RNA by intact nuclei, but in contrast to previous studies, they demonstrate that lower levels of ionic strength that are much closer to the physiological range may also strongly influence the synthesis of preribosomal RNA.

The RNA polymerase activity of intact nuclei exhibits a biphasic response to increasing ionic strength. The initial peak of stimulation reaches its maximum at an ionic strength equivalent to that of 40 mM (NH&S04; the second peak becomes apparent only at markedly higher ionic strengths. The overall pattern observed with intact nuclei appears to be the summation of separate effects on nucleolar and extranucleolar polymerase activities. Isolated nucleoli exhibit only a single peak of stimulation which corresponds to the one observed at low ionic strength with intact nuclei. The second peak of stimulation is probably of extranucleolar RNA synthesis; indeed, we have recently prepared an extranucleolar particulate RNA polymerase from sonically disrupted nuclei which is unaffected by the low ionic strengths that enhance nucleolar activity but is markedly stimulated by high ionic strengths in the range of 160400 mM (NHJS04.6 The results of base composition analysis, the sensitivity to actinomycin D, and the fact that isolated nucleoli exhibit only the single peak at a similar (NH&SO4 concentration confirm that the nuclear RNA synthesis stimulated by low ionic strength represents predominantly preribosomal RNA.

Stimulation of preribosomal RNA synthesis by ionic strength has not been observed in previous studies with isolated nuclei. In most of these studies (13-E), however, the effects of (NH&SO4 were examined in the presence of relatively high concentrations of Mn2+ which we have found to

6 J. D. Johnson and B. A. Jant, unpublished observations.

inhibit the RNA polymerase activity present at low ionic strength (Figs. 1 and 3). To observe the stimulation by low ionic strength it is necessary to maintain the divalent cation concentration below or close to the optimal level over the entire range of ionic strengths examined.

The range of ionic strength encompassing the entire peak of stimulation of pre-rRNA synthesis is relatively narrow and corresponds quite closely to the levels of ionic strength normally existing in the intracellular milieu. This suggests the possibility that intracellular ionic concentrations may play a significant role in the regulation of pre- rRNA synthesis in various physiological and pathological states. For example, many hormones have been found to enhance the synthesis of pre-rRNA and to raise the content of ribosomes in the cells of their target tissues (2). Despite much speculation and repeated efforts, however, there has as yet been no convincing demonstration of a direct effect of any hormone on nuclear RNA polymerase activity in mammalian systems (2, 38). Many of these hormones, such as cortisone, aldosterone, estrogens, androgens, and thy- roxine, have been found to influence ion movements and are thus capable of altering the intracellular ionic environment (39, 40). In view of the results of the present studies, it becomes more readily conceivable that hormonal influences on gene activity, spe- cifically on pre-rRNA synthesis, may be mediated by their effects on intracellular ion composition, a thesis previously pro- posed by Kroeger (41).

Recently Roeder and Rutter (42,43) have solubilized two distinct RNA polymerases from rat liver nuclei, one derived from nucleoli and the other from nucleoplasm. These soluble enzymes exhibit certain similarities to the particulate enzymes employed in our studies. In the presence of low ionic strength, e.g., about 40 mM (NHI)zSO+ the patterns of activation of the two soluble enzymes by divalent cations are very close to those which we observed for the nucleolar and extranucleolar RNA polymerase activities. Furthermore, the solubilized nucleoplasmic enzyme is stimulated by high ionic strength with an optimum between 120 and 150 mM (NH4)2-


SO+ and our particulate extranucleolar 9. NATR, K. G., CUTILLETTE, A. F., ZAK, R.,

RNA polymerase activity is maximally KOIDE, T., AND R.~BINOWITZ, M., Circ. Res.

stimulated at about 160 mM (NH,)$04.” 23, 451 (1968).

The solubilized nucleolar polymerase, how- 10. SOBEL, B. E., AND KIUFMAN, S., Arch. Bio-

ever, exhibits no stimulation by low ionic them. Biophys. 137, 469 (1970).

strengths between 0 and 50 mM (NH,)&304 11. THOMPSON, L. R., AND MCCARTHY, B. J.,

and is inhibit.ed by higher salt concentra- Biochem. Biophys. Res. Commun. 80. 166

tions. The assays were carried out, however, (1968).

with calf thymus DNA rather than endoge- 12. POOO, A. O., LITTAU, V. C., ALLFREY, V. G.,

AND MIRSKY, A. E., PTOC. Nat. Ad. Sci. nous DNA as the template. It may be, U.S.A. 67, 743 (1967). therefore, t,hat the stimulation of nucleolar 13. WIDNELL, C. C., AND TAT-I, J. R., Biochim. pre-rRNA synthesis by low concentrations Biophya. Acta 133, 478 (1966).

of (NH,)$Ok which we observed with intact 14. BLACKBURN, K. J., AND KLEMPERER, H. G.,

nuclei and nucleoli represents the unmasking Biochem. J. 192, 168 (1967).

for transcription of additional DNA cistrons 15. CHAMBON, P., RAMTJZ, M., M~NDEL, P., AND

which code for pre-rRNA. This difference DOLY, J., Biochim. Biophys. Acta 167, 594

between the solubilized and particulate (1968).

nuclear RXA polymerase activities serves to 16. MEISLER, A. I., AND TROPP, B. E., Biochim.

emphasize that although detailed under- Biophys. Acta 174, 476 (1969).

17. PEGG, A. E., AND KORNER, A., Arch. Biochem. standing of t,he mechanisms of transcription Biophys. 118, 362 (1967).

in eukargotic organisms requires the study of 18. WIDNELL, C. C., AND TATA, J. R., Biochem. J.

solubilized and purified enzymes, analysis 92, 313 (1964).

of regulat,orv phenomena mav require, in 19. MURAMATSU, M., SMETANA, K., AND BUSCH,

addition, the study and knowledge of ‘the properties of the intact organelle.

ACKNOWLEDGMENTS

We are indebted to Dr. Thomas S. Reese of the Laboratory of Neuroanatomical Sciences, Na- tional Inst,itute of Neurological Diseases and Stroke, for the electron microscopy of the nucleo- Iar preparations. We also express our appreciation to Esther Lewis, Lucille Wilfand, and John Kline for their excellent technical assistance.

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Effects of ionic strength on the RNA polymerase activities of isolated nuclei and nucleoli of rat...

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