a2p-distribution in oligonucleotides of rapidly ... · a2p-distribution in oligonucleotides of...

[CANCER RESEARCH 29, 1755--1762, October 1969]

a2p-Distribution in Oligonucleotides of Rapidly Sedimenting Nucleolar RNA's of Hepatomas and Normal Rat Liver

Ebrahim Yazdi, Tae Suk Ro-Choi, Joan Wikman, Yong C. Choi, and Harris Busch

Department of Pharmacology, Tumor By-Products Laboratory, Baylor College of Medicine, Houston, Texas 77025

SUMMARY

Nucleolar 28 S, 35 S, and 45 S RNA's were isolated from normal rat liver, Novikoff hepatoma ascites cells, and Morris hepatoma 9618A after short (60 min) and long (6 hr) labeling with orthophosphate-32p. After purification by sucrose density sedimentation, the 32p nucleotide composition of these RNA's was determined. The RNA was hydrolyzed to oligonucleotides with pancreatic ribonuclease. The distribution of radioactivity in the oligonucleotides was analyzed after they were separated by electrophoresis and chromatography.

In both the 60-min and the 6-hr labeling experiments, the distributions of the isotope in the oligonucleotides of nucleolar 28 S, 35 S, and 45 S RNA were virtually identical in any given tissue studied. The distributions of the isotope in the oligonucleotides in the nucleolar RNA's of normal liver were very similar in many respects to those in the tumors. However, in both the short and long labeling experiments, the isotope content of dinucleotides (mainly GC, the dinucleotide of guanylic and cytidylic acids, and GU, the dinucleotide of guanylic and uridylic acids) from both tumors was significantly higher than in the RNA of normal liver. The isotope content of the trinucleotides and tetranucleotides of the tumor RNA's were lower than those of the liver RNA's.

INTRODUCTION

The role of the nucleolus in the production of ribosomal RNA and possibly the whole ribosomal ribonucleoprotein complex has been extensively reviewed recently (5, 12, 17, 21). All of the studies made thus far support the concept that the initial nucleolar RNA product is a large molecule with a sedimentation coefficient of 45 S or greater (14, 15, 19). Studies on isolated nucleoli have shown that there is a progressive decrease in the sedimentation coefficient of the RNA from 45 S to 35 S to 28 S. Neither the number of RNA species in the nucleolar 28 S RNA (26) nor the composition of

I This work was supported by the Cancer Center Grant No. CA-10893-01, the American Cancer Society Grant No. P/339 D, and the Jane Coffin Childs Fund.

Received December 30, 1968; accepted June 5, 1969.

the nucleolar 28 S RNA have yet been clarified, partly because of the difficulties of fractionation of the high molecular weight nucleolar RNA and partly because of the relative insolubility of the GC-rich RNA which is the major com- ponent of nucleolar RNA. Recently, the suggestion has been made that nucleolar 28 S RNA contains both 18 S and 28 S ribosomal RNA (28).

As a result of the difficulties in fractionation and analysis of the high molecular weight nucleolar RNA's, comparative studies between the nucleolar RNA's of tumors and other tissues have been limited to studies on UVand 32p nucleotide compositions. In studies of this type made in this and other laboratories (6, 8, 9, 14-16, 18, 20, 27, 30), consistent differences have been found between the 32p nucleotide compositions of the tumor and nontumor tissues, particularly in the comparative studies on Morris hepatomas and normal and regenerating liver. The major difference is that in the tumor, the nucleolar 45 S RNA has a lower content of adenylic acid (AMP) and a higher content of uridylic (LIMP) and cytidylic acids (CMP) (4).

As an initial approach to the survey of larger oligonucleotides of nucleolar RNA of tumors and other tissues, the "mapping" procedure developed by Rushizky and Knight (23)appears to have considerable usefulness. This method has been applied by Jeanteur et al. (11) to the comparative analysis of nucleotides in the ribosomal and nucleolar RNA of HeLa cells and by Roberts and D'Ari (22) to the determination of the oligonucleotide frequencies of the ribosomal 18 S and 28 S RNA in comparison to those of the whole nuclear RNA's for Ehrlich ascites cells. The present study was designed to determine whether the low AMP and high CMP content of tumors after a short pulse (15 to 30 min) with 32 p would remain the same after long labeling (60 min to 6 hr) with orthophosphate -32 P. The results indicate that the differences between the 32p nucleotide composition of the tumor and liver nucleolar RNA's are consistent and suggest that differences in the specific activity of the four precursor nucleotide pools are not the major factor in the differences of 32p nucleotide composition of normal and tumor nucleolar RNA's. Another objective was to determine whether the oligonucleotide frequencies would be generally different in the nucleolar RNA of the tumors and the normal liver or whether the differences were restricted to a relatively small number of the oligonucleotides; the latter seems to be the case.

OCTOBER 1969 1755

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E. Yazdi, T. S. Ro-Choi, J. Wikman, Y. C. Choi, and H. Busch

MATERIALS AND METHODS

Male albino rats weighing 175-250 gm were obtained from the Cheek-Jones Co., Houston, Texas; they were fed Purina lab chow ad libitum. In the 60-rain labeling experiments, 2 mc of carrier-free orthophosphate-32P (Union Carbide Nuclear Co., Oak Ridge, Tenn.) in 0.2 ml saline solution brought to pH 7.0 with t N NaOH, were injected i.p. int~ each rat. Sixty rain later the rats were anesthetized with diethylether. The livers were perfused in situ with ice-cold 0.25 M sucrose. The excised livers were placed in ice-cold 0.25 M sucrose and transferred to a cold room (3-4~ In the 6-hr labeling experiments, 2 mc orthophosphate-a2P were injected i.p. into each rat. Three hours later an additional 2 mc of orthophosphate-32P were injected. Three hours after the second injection of 32p (6 hr after the first injection), the rats were sacrificed.

For Novikoff hepatoma ascites cells, approximately 3 • 107 cells were transplanted intraperitoneally into each animal. Six or 7 days after transplantation, rats were injected intraperitoneally with orthophosphate-a2P as described above and then sacrificed.

For Morris hepatoma 9618A, tumors were transplanted into both the right and left thigh muscles of Buffalo rats approximately one year prior to the experiments. Two mc of orthophospate-a2P were injected i.p. into each rat. Sixty rain later they were sacrificed, and their tumors were removed.

Isolation of Nuclei and Nucleoli. Nuclei were isolated by the modification of the previously employed Chauveau technic (3, 4, 17, 18, 26). The sonication procedure was employed for isolation of highly purified nucleoli (3, 17, 18).

Extraction and Purification of Nucleolar RNA. Purified nucleolar RNA was obtained by the method employing hot phenol-sodium dodecyl sulfate (17). The liver nucleolar RNA was centrifuged through a 10-50% sucrose gradient in an SW-25.3 rotor at 25,000 rpm for 20 hr at 4~ The larger amounts of tumor nucleolar RNA were centrifuged through a 5-40% sucrose gradient in an SW-27 rotor at 25,000 rpm for 16 hr at 4~ The sucrose gradients contained 0.1 M NaC1, 0.001 M ethylenediaminetetraacetate, and 0.01 M sodium acetate (pH 5.1). The gradients were fractionated in an ISCO gradient fractionator (ISCO, Lincoln, Nebraska).

Fractions corresponding to the rapidly sedimenting classes of RNA with approximate sedimentation coefficients of 28 S, 35 S, and 45 S were pooled and 0.5-1.0 mg carrier RNA (yeast RNA, Calbiochem, Los Angeles, Calif.) was added. The RNA was precipitated by adding 2.0-2.5 volumes of ethanol containing 2% potassium acetate and storing it a t -20~ overnight. Each sedimentation class of RNA was purified by 2 to 3 repeated sucrose density gradients until one symmetrical peak was obtained (27). For assay of radioactivity, 1 to 2 drops of 12 N perchloric acid (PCA) were added to each 0.25-ml fraction of sucrose gradient obtained from the ISCO fractionator, and each sample was heated at 70~ for 30 rain. Radioactivity was determined in a Packard liquid scintillation counter using the solvent system described by Bruno and Christian (2).

a2p Nucleotide Composition. Approximately 0.25 mg carrier RNA was added to a 25-/al aliquot (approximately 10,000 cpm) of RNA solution which was desalted on Sephadex G-25.

The RNA was precipitated by addition of ethanol containing 2% potassium acetate; it was then hydrolyzed with 50/al of 0.3 N KOH for 18 hr at 37~ The pH of the hydrolysate was adjusted to 2-3 with 1 N PCA at 0-2~ and centrifuged. The supernatant was removed, and its pH was adjusted to 7-7.5 with 1.0 N KOH. Potassium perchlorate was precipitated by repeated freezing and thawing, and it was removed by centrifugation. Separation of nucleotides was carried out by thin-layer electrophoresis at pH 3.15 (29). A 20 x 20 cm precoated cellulose sheet (Eastman Chromagram sheet #6065) with a fluorescent indicator was dipped into a solution of ammonium acetate (pH 3.15) prior to application of the sample and permitted to evaporate at room temperature for 30 rain; 10-20 /al of hydrolysate were then applied. Electro- phoresis was carried out for 1.5-2 hr at 700-800 volts and 15-20 ma in a Brinkmann Desaga chamber. Electrophoresis was terminated when UV light showed that the nucleotides were separated. The cellulose sheet was then dried, and the absorbing spots were cut out. The isotope content was determined in the scintillation counter.

Enzyme Digestion. To each purified 28 S, 35 S, and 45 S RNA, 4 mg carrier RNA (Yorula RNA, Calbiochem., Los Angeles, Calif.) were added. The final volume was 1-2 ml. These solutions were desalted by eluting with water on a Sephadex G-25 column in the cold room (22). The RNA- containing solutions were lyophilized, and the RNA was dissolved in 0.25 ml water. Ten #1 of 1.0 M sodium acetate buffer, pH 7.1, and 20 #1 of pancreatic RNase (500 units) were added to this solution. Digestion was carried out at 37~ for 20-24 hr. The solution of digested RNA was then concentrated to 50-100/al by an air jet.

Oligonueleotide Mapping Procedure. For mapping of digested RNA, the procedure of Rushizky and Knight (23) was used, i.e., paper electrophoresis at pH 2.7 (ammonium formate) in one dimension followed by paper chromatography in a 55:45 t-butanol:ammonium formate (pH 3.8) buffer, pH 4.7-4.8, in the other. Electrophoresis was carried out at pH 2.7 on Whatman 3MM paper (68 x 46 cm) for 17-20 hr with a potential of 6 volts/cm and a current of 10-20 ma. Electro- phoresis was terminated when a picric acid marker reached the buffer level in the anode chamber. For paper chromatography, the tank was completely saturated with the solvent, and the chromatogram was developed for 36-38 hr at room temperature. The papers were then dried, and the spots were located under UV light.

The spots were then cut out and eluted with 2-3 rill 0.01 N ttC1. To each sample, 10-20 ml of scintillating solution (2) were added. After thorough mixing, the radioactivity was counted in a Packard liquid scintillation counter.

RESULTS

Sucrose Density Gradient Sedimentation Profiles. Charts 1 and 2 show the gradient centrifugation pattern and radioactivity distribution in nucleolar RNA of hepatomas and normal rat liver after 60-rain and 6-hr labeling with orthophosphate-a2P respectively. The nucleolar RNA was separated into four major subfractions with sedimentation coefficients of

1756 CANCER RESEARCH VOL. 29



Oligonucleotides o f Nucleolar R N A

NOVI KOFF 28S 0.5F HEPATOMA /~35S ~ - ]5,000 0.4 ~- NUCLEOLAR / v l 14,000

I RNA i .L 4 S o.~ ~ I,Y"t "~s 1 ~,ooo

~ - i ~ -I~'~176176 0 , 1 , ,7 1,000

I (5%) 20 40 60 80 I00 120 ( 4 0 % ) MORRIS ',

~ 0.5r HEPATOMA 28S I0,000 ', ~ 9618A ~

0.4 NUCLEOLAR RNA 5S/", ~ 8,000

"~ OISF 4.7S L'i ~ ' 4 5S I , S ,O 0 0

"' 7A ~ <'<' 0.2 4,000 (5

~> o.; .-. z,ooo " i i i , o

I RAT L,V~R ~s < 0.41- NUCLEOLAR A35S 4,000

0 5 , s 5,000

0.2L 4-7s Jl; \/',,k 2,000 ,,ooo

(10%) I0 20 50 40 50 60(50~ FRACTIONS ( 0.25 ml )

Chart 1. Sucrose density gradient sedimentation profiles of nucleolar RNA of Novikoff hepatoma ascites cells, Morris hepatoma 9618A, and normal rat liver after 60-rain labeling with 2 mc orthophosphate-32p. For Novikoff hepatoma, the profile was obtained by ultracentrifugation of RNA in a linear sucrose density gradient (5-40%) in the rotor SW-27 (Spineo L) at 25,000 rpm at 4~C for 16 hr. Morris hepatoma and rat liver proffies were obtained by ultracentrifugation of RNA in a linear sucrose density ogradient (10-50%) in rotor SW 25.3 (Spinco L) at 25,000 rpm at 4 C for 20 hr. The solid lines present the absorbancy at 254 m/_t measured continuously. The dotted lines show the distribution of radioactivity measured in 0.25-ml fractions. The arrows show the directions of sedimentation. The approximate sedimentation coefficients are indicated.

approximately 4-7 S, 28 S, 35 S, and 45 S. The highest peaks were in the 28 S and 35 S regions.

The radioactivity distributions in nucleolar RNA's of Novikoff hepatoma, Morris hepatoma 9618A, and normal rat liver after 60-min or 6-hr labeling with or thophosphate-32p were very similar (Charts 1, 2). The three peaks of radioactivity coincided with the optical density peaks in the 28 S, 35 S, and 45 S regions.

Charts 3 and 4 show the purification of 28 S, 35 S, and 45 S RNA's of the liver and Novikoff hepatomas. Monodisperse peaks corresponding to the 28 S, 35 S, and 45 S RNA sedimentat ion classes were obtained after 2 or 3 purificatioh steps by repeated sucrose density gradient centrifugation (26). The purif ication of 28 S, 35 S, and 45 S RNA's for Morris hepa toma 9618A was essentially the same as for Novikoff hepa toma (Chart 4).

32p Nueleotide Compositions. The distr ibution of the isotope among the four nucleotides of nucleolar RNA's at 60 rain and 6 hr after injection of or thophosphate-32P is shown in Tables 1 and 2. In the tumors, the 32p nucleotide composi t ion in 60-min labeling experiments was not appreciably different from that of the 6-hr labeling experiments. In normal liver, however, there was a significant decrease in the isotope content o f AMP and UMP as well as a

0.5 NOVIKOFF 28S -----,, 30 ,000 1 ,ooo T

.,.r "~ 4 I0 ,000 t

, . ooo

t - -

O.5F RAT LIVER o~o ----" 0.4~ NUCLEOLAR "~35S

RNA

o.2p 4-7s J -

oO., i i i [

(IO%) IO

0 E

4000

3000

2000

I000

20 30 4 0 50 60(50%) TUBE NUMBER

Chart 2. Sucrose density gradient sedimentation profiles of nucleolar RNA of Novikoff hepatoma ascites cells and normal rat liver after 6-hr labeling with orthophosphate-32P. The profiles were obtained as described in Chart 1. Rats were injected with 2 mc 32p 3 hr and 6 hr prior to sacrifice.

RAT LIVER NUCLEOLAR RNA

~" 0.5

r~ ~ O A ~ 0.3 0.2

~0. I o <~ (10%) 8 16 24 32 40 48 56 (50*/.)

FRACTIONS (0.25 ml)

PURIFICATIONS FINAL PURIFICATIONS 0::~; F I R S T , 28S RNAI / 28S RNA

0.2: ! /' !

0.5 ~ , ' . . . . . ; . ;, . . . . ~" 0 4 ~ S RNA /; "'~ 35S RNA

m , i , ,i - - , j

0.4 45S RNA

o.2 ,: ~ ,: o.,' ", ~ . ~ ,

uOO, o) & '6 t4 32 40 4'8 ~$(50~..0~.~'8 .'6 z'4 s'2"--"I~\40 4'8 s~(sO~,o) FRACTIONS (0.25 ml)

Chart 3. Purification of 28 S, 35 S, and 45 S RNA's of rat liver nucleoli. Fractions taken from the shadowed portion of the original nucleolar RNA sedimentation pattern (top) were pooled and precipitated and recentrifuged two or three times in the presence of 0.5-1.0 mg carrier RNA. After each purification step, only the fractions corresponding to the shadowed zones were collected. Centrifugation was performed under the same conditions as described in Chart 1. The solid lines represent nucleolar RNA; the dotted lines correspond to carrier RNA. The arrow indicates the direction of sedimentation.

OCTOBER 1969 1757




NOVIKOFF HEPATOMA NUCLEOLAR RNA

N 2.0

15

0.5 ixi < (5"/.I 5 I0 15 20 25 (40*/ , )

FRACTIONS (I.0 rnl) 2.5 FIRST PURIFICATIONS

1.0 ,""

0.5 , _ j / \

2 .5 ' ' "~ ' '

2.0 35S RNA

F 1.5

~ 1.0

0.5 , / " \ "" \, m . i 'x < 2 . 5 I ' ' " . , ~ l I , [

2.0 45S RNA F 45S RNA

.:'",, I .'/'i 0 .5 / - / \

"- ; '-~ l + + 5 0 ) 5 I0 15 20 25(40%)(5%) ~) +0 15 20 25 ( 4 0 % )

FRACTIONS (l.O ml)

28S RNA

FINAL PURIFICATIONS

55S RNA

Table 1

A + U RNA AMP UMP GMP CMP G + C

Liver 28 S 22.4 + 0.3 20.4 + 0.4 33.3 + 0.2 23.8 + 0.2 0.75 35 S 21.0 + 0.7 19.5 + 0.2 35.5 + 0.6 23.8 + 0.8 0.68 45 S 21.1 + 1.0 18.4 + 0.1 35.5 + 1.0 25.2 + 0.6 0.65

Novikoff hepatoma 28 S 13.8 + 0.3 21.0 + 0.2 33.6 + 0.7 31.7 + 0.6 0.53 35 S 13.7 + 0.4 20.9 + 0.6 33.5 + 0.3 31.3 + 0.4 0.53 45 S 13.5 + 0.7 20.6 + 0.6 33.6 + 0.2 31.7 + 0.3 0.52

Morris hepatoma 9618A 28 S 15.8 + 0.8 18.4 + 0.1 35.3 + 0.6 30.3 + 0.8 0.52 35 S 16.6 + 0.6 18.7 + 0.7 34.7 + 0.1 30.1 + 0.1 0.54 45 S 15.7 + 0.2 19.4 + 0.2 33.2 + 0.3 31.6 + 0.2 0.54

Nucleotide composition of rapidly sedimenting nucleolar RNA's from normal rat liver, Novikoff hepatoma, and Morris hepatoma 9618A after 60-min labeling with orthophosphate-a2P. Each rat received 2 mc of orthophosphate-32P i.p. 60 min before it was sacrificed. The values are percentages of total radioactivity in the four nucleotides. Standard errors were calculated by the following equation: S.E. = N/2(x-x~2/n(n-1). The values are averages of 3 experiments for each tissue. AMP, adenylic acid; UMP, uridylic acid; GMP, guanylic acid; CMP, cytidylic acid; A + U/G + C, ratio of adenylic acid and uridylic acid to guanylic acid and cytidylic acid.

Chart 4. Purification of 28 S, 35 S, and 45 S nucleolar RNA's of Novikoff hepatoma ascites ceUs. Fractions taken from the shadowed portion of the original nucleolar RNA sedimentation pattern (top) were pooled and precipitated and recentrifuged two or three times in the presence of 0.5-1.0 mg carrier RNA. After each purification step, only the fractions corresponding to the shadowed zones were collected. Centrifugation was performed under the same conditions as described in Chart 1. The solid lines represent nucleolar RNA; the dotted lines correspond to carrier RNA. The arrow indicates the direction of sedimentation.

concomitant increase in the isotope content of GMP and CMP. The percentage of radioactivity in AMP in all three

sedimenting classes of nucleolar RNA's, e.g., 28 S, 35 S, and 45 S from Novikoff hepatoma and Morris hepatoma 9618A, was lower than that of normal liver. These findings are in agreement with previous reports (4, 6, 14, 15, 18) on the differences of nucleotide composit ions of tumors and liver nucleolar RNA's after short 32p pulses.

The persistence of lower AMP and higher CMP content of tumor nucleolar RNA's after the long labeling periods of 60 min and 6 hr suggests that the differences in the specific activity of the four precursor nucleotide pools is not the major factor in the differences of 32p nucleotide composi t ion between normal and tumor tissue.

Mapping of Products Obtained from Enzymatic Digestion of RNA's

Nucleotide Frequencies of Ribosomal Precursor RNA's. Tables 3 and 4 show the distributions of radioactivity in

Table 2

A + U RNA AMP UMP GMP CMP G + C

Liver 28 S 19.0 + 0.2 16.0 + 0.2 36.6 + 0.8 28.5 + 0.7 0.54 35 S 17.5 + 0.1 15.3 + 0.3 38.4 + 0.1 28.7 + 0.3 0.49 45 S 18.3 + 0.3 16.2 + 0.7 36.7 + 0.0 28.6 + 0.2 0.53

Novikoff hepatoma 28 S 14.4 + 0.2 20.2 + 0.5 34.5 + 0.2 31.9 + 0.3 0.52 35 S 14.4 + 0.2 18.8 + 0.5 34.0 + 0.1 32.0 + 0.2 0.50 45 S 14.0 + 0.2 19.5 + 0.3 34.6 + 0.3 32.2 + 0.2 0.50

32p nucleotide composition of rapidly sedimenting nucleolar RNA's from normal rat liver and Novikoff hepatoma ascites cells after 6-hr labeling with orthophosphate -32p. Each rat received 2 mc of orthophosphate-32P i.p. 3 hr and 6 hr before it was sacrificed. The values are percentages of total radioactivity in the four nucleotides. Standard errors were calculated by the following equation: S.E. = N/Y-(x-x--)2/n(n-1) �9 The values are averages of 3 experiments for each tissue. AMP, adenylic acid; UMP, uridylic acid; GMP, guanylic acid; CMP, cytidylic acid: A + U/G + C, ratio of adenylic acid and uridylic acid to guanylic acid and cy tidylic acid.

oligonucleotides of nucleolar RNA's of rat liver, Novikoff hepatoma, and Morris hepatoma 9618A after a 60-min labeling period with orthophosphate-a2P. The composit ions of each oligonucleotide obtained from the pancreatic RNase digest were previously defined in these maps by Rushizky and Knight (23) and Roberts and D'Ari (22). The distribution of the digestion products and the radioactive spots was virtually




Oligonucleo tides of" Nucleolar RNA

Table 3

Normal rat liver Novikoff hepatoma Morris hepatoma (Oligo) Nucleotides a 28 S b 35 S 45 S 28 S 35 S 45 S 28 S 35 S 45 S

C 28.8 + 0.7 28.5 -+ 0.2 27.6 + 0.1 28.1 + 0.5 26.6 + 0.3 26.1 + 0.5 28.8 + 0.3 28.6 + 0.4 28.0 + 0.5 U 13.8 + 0.8 14.6 - 0.3 15.9 + 0.4 12.8 + 0.3 12.9 + 0.1 15.0 + 0.5 14.7 + 0.2 14.2 + 0.2 14.5 + 0.3 Pseudo-U 1.7 + 0.0 1.3 + 0.0 0.9 + 0.1 0.5 + 0.0 0.5 + 0.0 0.5 + 0.0 0.8 + 0.0 0.6 + 0.1 0.6 + 0.1

44.3 + 0.5 44.4 + 0.7 44.4 + 0.6 41.4 40.0 41.6 44.3 + 0.4 4~4 + 0.3 43.1 + 0.3

AC 5.1 +- 0.0 5.0 + 0.1 5.0 + 0.0 5.5 + 0.3 5.2 + 0.1 4.9 + 0.3 5.2 + 0.1 5.3 + 0.1 5.1.-+ 0.2 GC 12.2 + 0.6 12.3 + 0.5 12.9 + 0.5 15.1 + 0.3 15.5 4_ 0.4 15.6 + 0.3 18.3 + 0.2 18.5 + 0.2 19.0 + 0.2 AU 2.8 + 0.0 2.7 + 0.1 2.8 + 0.0 3.1 + 0.2 2.4 + 0.1 2.8 + 0.3 1.9 + 0.1 1.9 + 0.0 1.8 + 0.1 GU 4.4 + 0.3 4.8 + 0.0 4.3 + 0.0 9.7 + 0.2 9.1 + 0.3 9.8 + 0.4 6.3 + 0.1 6.4 + 0.1 6.4 + 0.2

24.5 + 0.2 24.8 + 0.4 25.0 + 1.4 33.4 32.2 33.2 31.7 + 0.2 32.1 + 0.3 32.4 + 0.3

AAC 3.1 + 0.1 3.2 + 0.3 3.1 + 0.1 2.2 +- 0.3 2.1 + 0.0 1.8 + 0.1 2.0 + 0.1 2.0 + 0.0 1.8 + 0.1 (AG~ 7.4 + 0.0 8.1 -+-+ 0.5 8.3 + 0.5 6.4 -+ 0.1 6.5 + 0.2 5.5 + 0.2 7.5 + 0.1 7.2 + 0.1 7.3 + 0.1 GGC/A3U 2.3 + 0.1 2.5 + 0.2 1.9 + 0.0 3.0 + 0.1 3.5 + 0.3 3.2 --+ 0.3 2.5 + 0.1 2.2 + 0.1 2.4 + 0.1 AAU 2.2 +- 0.1 2.0 + 0.I 2.4 + 0.0 1.3 + 0.3 2.2 + 0.1 1.6 + 0.4 1.5 + 0.1 1.7 + 0.0 1.5 + 0.1 (AG)U 3.2 + 0.0 2.9 + 0.3 3.1 + 0.3 3.0 + 0.2 3.7 + 0.2 3.2 + 0.2 2.2 + 0.1 2.7 + 0.1 2.5 + 0.1 GGU 1.1 + 0.1 1.8 + 0.2 1.4 --- 0.2 3.0 + 0.2 2.2 --+ 0.1 3.1 + 0.1 1.0 +- 0.1 1.0 + 0.1 0.9 + 0.0

19.3 -+ 0.3 20.5 + 0.1 20.2 + 0.6 18.9 20.2 18.4 16.7 + 0.1 16.9 + 0.1 16.4 + 0.I

A3C 1.7 + 0.0 1.6 + 0.1 1.2 + 0.1 1.7 + 0.3 1.2 + 0.1 0.8 + 0.3 1.1 + 0.1 1.4 + 0.1 1.1 + 0.1 (A2G)C 4.9 + 0.0 4.8 + 0.1 4.5 + 0.2 2.5 +- 0.0 1.7 + 0.1 1.7 + 0.1 2.6 + 0.1 2.6 + 0.1 3.0 + 0.1 (AG2)C 2.5 -I- 0.2 1.5 -I- 0.2 1.4 + 0.2 1.8 + 0.0 2.5 --- 0.0 1.6 + 0.1 1.4 +- 0.1 1.1 + 0.1 1.1 + 0.2 (A2G)U 1.6 + 0.2 1.4 -+ 0.1 2.4 + 0.1 1.6 + 0.0 1.6 + 0.0 2.4 + 0.1 1.4 + 0.1 1.5 + 0.1 2.3 + 0.1 (AG2)U 1.0 + 0.0 0.8 -+ 0.0 1.2 + 0.0 0.7 + 0.0 0.9 + 0.0 1.2 + 0.0 0.6 + 0.1 0.8 + 0.1 0.6 + 0.1

11.7 -+ 0. t 10.1 -+ 0.0 10.7 + 0.0 8.3 8.0 7.7 7.1 -+ 0.3 7.4 + 0.1 8.1 + 0.2

Radioactivity distribution in mono- and oligonucleotides of nucleolar RNA's after 60-min labeling with orthophosphate-32p. The values for each sequence are the percentages of the total radioactivity from each RNA found in 18 eluted spots.

aAbbreviations are those used by Rushizky and Knight (23). bEach value represents an average of 3 to 6 determinations from separate experiments. The standard errors were calculated by file following

equation: S.E. = ~ (xL'~)2/n(n-1).

identical to that reported by Rushizky and Knight (23) z. [The abbreviations in this paper are those used by Rushizky and Knight (23).] Approximately 72-75% of the radioactivity of each RNA was found in 18 oligonucleotides. The RNA core "resis tant" to pancreatic RNase remained at the origin. This fraction along with larger oligonucleotides which do not emerge from the origin accounted for an average of 27.0 + 0.50 and 28.0 +- 0.70 of the total radioactivity for liver and tumor RNA's respectively.

The distribution of radioactivity among mono- and oligonucleotides of nucleolar RNA's from rat liver and Novikoff hepa toma after 6-hr labeling with orthophospate-3ZP is shown in Table 4.

2The A3U tetranucleotide was not always separated from trinucleotide GGC spots, and an extra spot was found which corresponds to that identified by Roberts and D'Ari (22) and Ingrain (10) as pseudouridylic acid. This finding of pseudo-U in high molecular weight RNA's agrees with the results of Dunn (7), Amaldi and Attardi (1), Roberts and D'Ari (22), and Jeanteuret al. (11).

In the experiments at both 60 min and 6 hours, the nucleotide frequencies for the 28 S, 35 S, and 45 S nucleolar RNA's were virtually identical for a given tissue.

The remarkable similarity in the isotope content of oligonucleotides of nucleolar 28 S, 35 S, and 45 S RNA's in all three types of tissues studied provides evidence that these three sedimentat ion classes of nucleolar RNA's contain species of ribosomal precursor RNA with very similar, or perhaps even identical, primary structure; these RNA's may be the oligomers of a c o m m o n polynucleot ide structure. The minor differences between the isotope contents of the oligonucleotides of nucleolar 28 S, 35 S, and 45 S RNA may result from elimination of segments of RNA from the large molecules during conversion of 45 S to 35 S and 35 S to 28 S RNA. Also, it is possible that these minor differences are due to the l imitation of the method . Similar minor variations have been reported in oligonucleotides of 28 S, 35 S, and 45 S RNA from Ehrlich ascites tumor (22).

The Effect of Time of Labeling on the Distribution of Radioactivity in Mono- and Oligonucleotides o f Nucleolar RNA's. The isotope contents of oligonucleotides of liver nucleolar RNA's after a 60-min labeling period are similar to those after a 6-hr labeling period (Tables 3, 4). Significant

OCTOBER 1969 1759



E. Yazdi, T. S. Ro-Choi, J. Wikman, Y. C Choi, and H. Busch

Table 4

Normal rat liver Novikoff hepatoma (Oligo) Nucleotides a 28 S c 35 S 45 S 28 S 35 S 45 S

C 26.7 + 0.7 27.8 + 0.5 27.7 + 0.5 28.7 + 0.5 29.0 + 0.2 27.7 + 0.4 U 13.4 + 0.6 13.1 + 0.3 12.6 + 0.4 13.6 + 0.4 13.2 + 0.2 13.7 + 0.3 Pseudo-'U 1.0 + 0.1 0.6 + 0.2 1.3 + 0.1 0.8 + 0.0 0.7 + 0.0 0.8 + 0.1

41.1 41.5 41.7 43.1 42.9 42.2

AC 4.3 + 0.1 4.3 + 0.1 4.6 + 0.3 4.2 + 0.1 3.9 + 0.2 4.4 + 0.1 13.2 + 0.0 14.0 + 0.0 13.5 + 0.2 14.4 + 0.2 14.6 + 0.3 15.0 + 0.1

AU 2.5 + 0.1 2.5 + 0.2 3.1 + 0.2 2.9 + 0.1 2.7 + 0.1 2.9 + 0.2 GU 5.8 + 0.2 5.9 + 0.4 5.8 + 0.2 9.3 + 0.2 9.4 + 0.0 9.8 + 0.1

25.8 26.7 27.0 30.8 30.6 32.1

AAC 2.7 + 0.1 2.5 + 0.3 2.8 + 0.2 1.8 + 0.1 1.7 + 0.1 1.7 + 0.1 (AG)C 6.5 + 0.1 6.5 + 0.2 5.6 + 0.1 5.0 + 0.1 5.5 + 0.2 4.9 + 0.1 GGC/A3U 5.2 + 0.4 5.0 + 0.2 4.6 + 0.3 3.4 + 0.5 3.7 + 0.4 4.4 + 0.2 AAU 1.8 + 0.1 2.1 + 0.2 1.5 + 0.1 1.4 + 0.1 1.5 + 0.1 1.3 + 0.1 (AG)U 3.8 + 0.2 3.0 + 0.5 3.6 -+ 0.2 2.7 + 0.3 3.4 + 0.2 3.6 + 0.3 GGU 2.6 + 0.2 2.1 + 0.4 2.7 + 0.2 3.1 + 0.1 2.7 + 0.2 2.6 + 0.2

22.0 21.2 20.8 17.4 19.5 18.5

AaC 1.3 + 0.1 1.5 + 0.2 1.5 + 0.1 1.4 -+ 0.2 1.3 + 0.1 0.9 + 0.1 (AAG)C 2.8 + 0.2 2.9 + 0.2 2.9 + 0.1 2.4 + 0.2 2.6 + 0.1 1.7 + 0.1 (AGG)C 3.1 + 0.2 2.6 + 0.4 2.0 + 0.2 1.4 + 0.1 1.6 + 0.2 1.5 + 0.1 (AAG)U 2.7 + 0.2 2.2 + 0.1 2.8 + 0.0 1.6 + 0.2 1.4 + 0.1 1.6 + 0.1 (AGG)U 1.2 +-- 0.2 1.3 + 0.2 1.7 + 0.3 1.4 + 0.1 1.0 + 0.1 1.0 + 0.0

11.2 10.5 10.9 8.2 7.9 6.7

Radioactivity distribution in mono- and oligonucleotides of nucleolar RNA's after 6-hr labeling with orthophosphate-a2P. The values for each sequence are the percentages of the total radioactivity from each RNA found in 18 eluted spots.

aThe abbreviations are those used by Rushizky and Knight (23). beach value represents an average of 4 to 8 determinations from separate experiments. The standard errors were calculated by the.

following equation: S.E. = V / Y~ (• _~)2/n(n_ l).

differences were found in the isotope content of U, GU, (AG)C, and (A2 G)C after 60-rain and 6-hr labeling periods.

On the other hand, in the Novikoff hepatoma the isotope contents of the oligonucleotides of nucleolar RNA's obtained after a 60-rain labeling period were not appreciably different from those obtained after 6-hr labeling with orthophosphate-32p (Tables 3, 4).

Comparative Analysis of Oligonucleotides of Tumor Nueleolar RNA's with Those of Liver Nucleolar RNA's. Although the isotope contents of oligonucleotides of 28 S, 35 S, and 45 S nucleolar RNA's from rat liver, Novikoff hepatoma, and Morris hepatoma 9618A are similar for a given tissue (Tables 3, 4), there are dissimilarities between the values for the tumors and normal liver. Significant differences were found between both tumors and the liver with respect to GC and GU (P < 0.001). Lesser but significant differences were found for AAC and (A2G)C ( P < 0.01).

On the other hand, no significant differences between the tumors and normal liver were found for some oligonucleotide frequencies, i.e., for C, U, AC, AU, (AG)U, A2 U, G2 U, A3 C, (AG2)C, (A2 G)U, and (AG2)U.

DISCUSSION

Although differences in nucleolar RNA's of tumors and other tissues will only be completely defined when the nucleotide sequences and configurations of these RNA's are established, at present there are considerable obstacles to fract ionat ion of the high molecular weight nucleolar RNA's and the deter- ruination of the sequences of such large molecules. The determination of partial oligonucleotide sequences is an intermediate step for studies on the structure of the very high molecular weight RNA of mammalian cells (1, 11, 22). Accordingly, the present studies are a step forward, but they only provide a partial answer to the structural differences of nucleolar RNA's of tumors and other tissues.

Since the pyrimidine contents of the rapidly labeled nucleolar RNA's in tumors are higher than those in liver, as found in 32p nucleotide composit ions (4, 13-16, 18, 26), the distribution of radioactivity among the products of pancreatic RNase digests of tumors and liver should be different. I f the differences were randomized, there should be more of the isotope in the small oligonucleotides of the RNA's of tumors




than in the liver RNA's. Although more isotope was found in the dinucleotides GC and GU of the tumors, the differences in the distr ibution of radioactivity among oligonucleotides of tumors and liver are not completely randomized. No significant differences were found between the livers and tumors for 32p distributions in the mononucleot ides C and U, the dinucleotides AU and AC, the trinucleotides A2 U, (AG)U, and G 2U, and the tetranucleotides A3C, (AG2)C, (AG2)U, and (A2G)U.

The differences in 32p content of the oligonucleotides of the tumors and liver cannot simply reflect pool differences in precursor nucleotide triphosphates. In sequences such as GU and AU, the isotope in the UTP pool is a common precursor of the isotope in the GU and AU; the percentage of isotope in AU was not significantly different in the tumors and the livers. Accordingly, the ratios of isotope in GU/AU reflects the ratio of the number of these dinucleotides in the labeled RNA molecules. Similarly, labeled CTP is a common precursor of the dinucleotides GC and AC. The percentage of isotope in AC was not significantly different in the tumors and the livers. Accordingly, the ratio of isotope in GC/AC reflects the ratio of the number of these dinucleotides in the labeled RNA molecules. Thus, these differences in the GC and GU contents of the tumors and the liver reflect sequential differences in these RNA molecules,3.

Al though these data extend the previous findings which suggest that the nucleolar gene readouts are significantly different in tumors and other tissues, they do not permit a precise analysis of differences with respect to the structure of the high molecular weight nucleolar RNA's. Even if it were assumed that the nucleolar 45 S RNA was a single molecular species, the problems of defining the total sequence of nucleotides are of considerable magnitude. One important aspect o f the present data is the finding o f many similarities of oligonucleotide composi t ions of tumors and liver nucleolar RNA's. Accordingly, analysis of partial sequences of nucleolar RNA may be helpful in defining differences in the RNA's of the tumors and normal liver. Studies on partial hydrolysis of nucleolar RNA's with limiting concentrat ions of RNases are in progress in this laboratory.

ACKNOWLEDGMENTS

The authors wish to express their appreciation to Dr. H. P. Morris, formerly of the Nutrition and Carcinogenesis Section, National Cancer Institute, NIH, Bethesda, Maryland, and now at Howard University, Washington, D. C., for his generous supply of Morris hepatoma 9618A. We also wish to thank Mrs. Rose K. Busch for her excellent work in transplantation of the tumors.

3One possibility considered for the differences in distribution in oligonucleotides of tumors and liver is that one or more of the nucleotides might be methylated differently. Although this possibility has not been ruled out in these studies, the nucleotide most frequently methylated is guanylic acid, and usually only approximately l f4 of the total nucleotides are methylated in these RNA's. However, the mobilities of oligonucleotides that contained methylated guanylic acid are not remarkably different from those of the nonmethylated analogs, and in these studies no marked differences in the spots were found in the maps in tumors and liver.

Oligonucleotides o f Nucleolar R N A

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1969;29:1755-1762. Cancer Res Ebrahim Yazdi, Tae Suk Ro-Choi, Joan Wikman, et al. Nucleolar RNA's of Hepatomas and Normal Rat Liver

P-Distribution in Oligonucleotides of Rapidly Sedimenting32

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