a2p-distribution in oligonucleotides of rapidly ... a2p-distribution in oligonucleotides of rapidly
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[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
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 signifi- cantly 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.
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 oligo- nucleotide 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 com- position 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 oligo- nucleotides; the latter seems to be the case.
OCTOBER 1969 1755
Research. on May 17, 2020. © 1969 American Association for Cancercancerres.aacrjournals.org Downloaded from
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 intraperi- toneally 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