- Email: [email protected]
Isolation of nucleoli from rat liver in the presence of magnesium ions
Experimental Cell Research:7 I (1972) 65-74
ISOLATION OF NUCLEOLI FROM RAT LIVER IN THE PRESENCE OF MAGNESIUM IONS T. HIGASHINAKAGAWA’*,
M. MURAMATSU’**
and H. SUGAN02
Departments of ‘Chemistry and 2Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, Tokyo, Japan
SUMMARY A procedure for the isolation of nucleoli from rat liver in the presence of Mg2+ ions was developed. Addition of 0.05 mM MgC& to the 0.34 M sucrose in which isolated nuclei were suspended for sonication was found to protect nucleoli when nuclei prepared with 2.3 M sucrose containing 10 mM MgCl, were disrupted completely by sonication. The nucleolar preparation possesses almost identical properties with those of nucleoli prepared by the conventional Ca2+-method in the following points: (1) morphological characteristics as revealed by light and electron microscopy; (2) RNA/DNA ratio of around 1.3; (3) sedimentation profiles and labeling patterns of nucleolar RNA; (4) polyacrylamide gel electrophoresis of acetic acid soluble proteins. Furthermore, Mg2+-nucleoli showed a higher RNA synthetic activity than Ca2+-nucleoli. In addition, this method has proved to be advantageous in the case of regenerating liver in which nuclei and nucleoli are somewhat more fragile to sonication.
Isolation of nucleoli from rat liver cells has the aid of Mg2+ ions and spermine, by which been reported by Muramatsu et al. [l], almost identical nucleolar preparations were where 3.3 mM Ca2+ ions were added in the obtained with regard to morphological criinitial homogenization medium. In view of teria. The nucleolar RNA extracted from the inhibitory effects of Ca2+ ions and the these preparations was almost indistinguishstimulatory effects of Mg2+ ions on some able from that extracted from Ca2+-nucleoli. enzymatic activities [2, 31 and the ability of However, the addition of spermine may not Mg2+ ions to preserve the integrity of ribo- be suitable for detailed biochemical analyses nucleoprotein particles [4], it is desirable of various nucleolar components, such as to have an alternative procedure for the ribonucleoproteins and enzymes, because of isolation of nucleoli that utilizes Mg2+ ions the biological activities of polyamines [6, 71. rather than Ca2+ions. During the course of studies on the nuBusch et al. [5] developed a procedure for cleolar ribonucleoprotein particles, we have the isolation of nucleoli from rat liver with developed a procedure for the isolation of nucleoli with Mg2+ ions without utilizing * Present address: Mitsubishi-Kasei Institute of Life Sciences, c/o Central Research Laboratory, Mitother additives including spermine. This subishi Chemical Industries Limited, 290 Hisamotopaper describes the isolation procedure and kamoicho, Kawasaki, Japan, * * Present address: Department of Biochemistry, some properties of the isolated nucleoli preTokushima University School of Medicine, Tokupared in this manner. shima, Japan. 5-721803
Exptl Cell Res 71
66
T. Higashinakagawa et al.
MATERIALS AND METHODS Isolation of nuclei and nucleoli The procedure was essentially the same as that of Caz+-method [1] except that the CaZ+ ions were replaced with Mg2+ ions at a modified concentration. All operations were carried out at 2-4”C. The perfused livers were weighed, minced and homogenized with three up and down strokes in 10 vol of 2.3 M sucrose-10 mM MgCI, in a loosely fitting PotterElvehiem type homogenizer with a Teflon pestle. The homogenate was filtered through four layers of cheese cloth and centrifuged down at 40 000 g for 1 h. The nuclear pellet was suspended in 0.34 M sucrose -0.05 mM MgC12, gently homogenized and sonicated in 20 ml batches in a Kubota Ultra Sonic Generator, Model KMS-250 (10 kc/set) at an output of 200 W for 45 to 60 sec. Thesonicatewasrapidly checked for the extent of the disruption of nuclei under a light microscope. When virtually all the nuclei were destroyed, the sonicate was underlaved with 20 ml of 0.88 M sucrose-O.05mM M&l., and centrifuged at 2 000 g for 20 min in a refrigerated centrifuge. The supernatant was discarded and the inside of the centrifuge tube was wiped well with a sheet of clean tissue paper. The resulting pellet contained purified nucleoli. Isolation of nucleoli from regenerating rat liver could be performed in a similar manner, although a shorter sonication time, approx. 20 set, was found to be sufficient. In these procedures the concentration of MgCl, in the initial homogenizing medium (10 mM) and the sonication medium (0.05 mM) was found to be critical for the effective disruption of nuclei and for the maximal yield of nucleoli.
Determination of DNA, RNA and protein DNA was determined with Burton’s procedure [8], and RNA was determined by the method of Fleck & Munro [9]. Determination of protein was performed according to the method of Lowry et al. [lo] with bovine serum albumin as standard.
RNA extraction and sedimentation analysis RNA was extracted from the nucleolar pellet with SDS-hot phenol procedure and subjected to sedimentation analysis as described previously [ll].
Polyacrylamide gel electrophoresis of nucleolar acetic acid-soluble proteins Nucleolar preparations obtained with either Ca2+or MaZ+-method were suspended in 67 % acetic acid and ailowed to stand with occasional shaking in the cold overnight. The suspension was centrifuged at 20 000 g fo; 20 min at O’C and the supematant was dialysed against 6 M urea, 15 % sucrose. Fifty to 100 pg of proteins were electrophoresed on a 15 % polyacrylamide gel containing 6 M urea. Electrophoresis was conducted under a constant current of 1 mA/column for approx. 15 h in the cold using Exptl Cell Res 71
glycine-acetate buffer containing 6 M urea (pH 4.0). The gels were stained with 1 % amido-schwarz 1OB in 10 % acetic acid, destained by standing in 10% acetic acid. In order to compare the proteins of Ma2+-nucleoli with those of Ca2+-nucleoli. the splitgei technique [12] was employed. In this-procedure the two protein samples were electrophoresed in a single gel column and were compared in a more direct and rigorous manner.
Electron microscopy Electron microscopic observations of the isolated nuclear and nucleolar preparations were performed as described previously [13].
Assay of RNA synthetic activity The activity of the isolated nucleoli to incorporate [‘“Cl UMP into acid-insoluble fraction was measured as described previously [ll].
RESULTS Concentrations of magnesium chloride
In the present experiment, a variety of concentration of MgCl, in the initial hypertonic sucrosemedium and in the sonication medium was examined. In general, Mg2+ ions seemed to render the nuclei resistant to sonication. The combination of 2.3 M sucrose- 2 10 mM MgCl, and 0.34 M - 1 0.05 mM MgCl, gave the most satisfactory results. Addition of more than 0.1 mM MgCl, to the sonication medium made the nuclei too hard to be broken within 1 or 2 min, and the final nucleolar preparation was grossly contaminated with unbroken nuclei. On the other hand when no Mg2+ ions were included in the sonication medium, the nucleoli were not sufficiently resistant to the sonication resulting in a lower recovery. Nucleolar preparations from regenerating rat liver were obtained similarly but in this case sonication of only 20 set or so was found to be sufficient to destroy all the nuclei and release the nucleoli. Sonication for a longer period resulted in the disintegration of hypertrophied nucleoli of regenerating liver.
Tion of nucleoli
67
Fig. I. Rat liver nuclei isolated with 10 mM MgCl, in the homogenization medium, staLinedwith azure C. Fig. 2. Higher magnification of fig. 1.
Exptl Cell Res 71
68
T. Higashinakagawa et al.
Fig. 3. Rat liver nucleoli isolated from nuclei of fig. 1 by sonicating fhe.m in 0.34 M sucrose MgCl,, stained with azure C. The blurred spots are in fact nucleoh shghtly out of focus. Fig. 4. Higher magnification of fig. 4.
Exptl Cell Res 71
containing 0.05 mM
Isolation of nucleoli 69
Fig. 5. Nuclei isolated from the 18-h regenerating rat liver with 10 mM MgCI, in the hor nogenization nTedium, stained with Azure C. Fig. 6. Nucleoli isolated from nuclei of fig. 4 by sonicating them in 0.34 M sucrose contai ining 0.05 mM MgCL stained with Azure C. Some of the nucleoli are shown as blurred spots because they are out of focus.
Exptl Cell Res 71
70
T. Higashinakagawa et al.
Fig. 7. Electron micrograph of rat liver nuclei isolated with Mg2+-method. x 3 300. Fig. 8. Electron micrograph of the isolated nucleolus of rat liver. Note the granular ribonucAeoprotein strucl :ures within the nucleolus. x 40 000.
Exptl Cell Res 71
Isolation of nucleoli
71
Morphology of isolated nuclei and nucleoli
The purity of the nuclear and nucleolar preparations is shown in figs l-8. The nuclear preparation showslittle cytoplasmic contaminations (figs 1, 2, 5, 7) and retains nucleolar structure which is well stained with Azure C (figs 1, 2, 5). The nucleolar preparations contain very little unbroken nuclei nor aggregated chromatin (figs 3, 4, 6). The nucleolar fine structures are presented in fig. 8. As already known, the ribosome-like granular structures are seen [14]. Figs 5 and 6 represent the nuclear and nucleolar preparations isolated from 18 h-regenerating rat liver with the present procedure. The large size and somewhat irregular shape of the nucleoli characteristic of regenerating liver can be seen. Composition of isolated nucleoli
The amounts of DNA, RNA and protein were determined for the nucleoli isolated with Mg2+-procedure. The results are presented in table 1. The RNA/DNA ratio is around 1.3, which is a little higher than that of Ca2+-nucleoli (1.02) [I].
TOP
BOTTOM
Fig. 9. Abscissa: tube number; ordinate: (left) Azs4; (right) cpm. -, Azs4; 0 - 0, radioactivity. Sedimentation profile of RNA extracted from the nucleoli isolated with Mg2+-method. Rats were injected intravenously 50 ,uCi per rat of aH-orotic acid 10 min prior to sacrifice. Nucleoli were isolated with Mg2+-method as described in Materials and Methods. RNA was extracted from the nucleolar pellet with SDS-hot phenol procedure and placed on a lo-40 % (w/w) sucrose gradient buffered in 0.01 M Naacetate, 0.1 M NaCl, 1 mM EDTA (pH 5.1) and centrifuged in a SW 25.3 rotor of Spinco L2 Ultracentrifuge at 24 500 rpm for 20 h at 4°C. The gradient was fractionated with the aid of an ISCO gradient fractionater, Model D, with an automatic optical density recorder at 254 nm. Each fraction was assayed for radioactivity in a liquid scintillation spectrometer as described previously [12].
In fig. 9 is shown the sedimentation profile of RNA extracted from Mg2+-nucleoli. The optical density profile clearly shows the existence of main peaks of 45S, 35S, 28 S and
4-6s RNAs and some other minor peaks, quite similar to the profile of RNA from Ca2+-nucleoli. It is also clear from the labeling pattern that at 10 min labeling with 3Herotic acid, the radioactivity was distributed exclusively over 45s region, as was also the case with Ca2+-nucleoli [15]. These data clearly indicate the intactness of the RNA of the nucleoli prepared with Mg2+-method.
Table 1. Composition of the nucleoli isolated
Acetic acid-soluble proteins of the nucleoli
with Mg2f-method
Proteins were extracted from the isolated nucleoli with 67 % acetic acid and electrophoresed on the polyacrylamide gel. To compare the proteins of Mg2+-nucleoli with those of Ca2+-nucleoli, proteins were extracted from Ca2+-nucleoli and electrophoresed in the split-gel column. No appreciable difference was detected between the two protein samples as shown in fig. 10.
Integrity of nucleolar RNA
RNA
Protein
Ewt (A
DNA
old
ocd
21
448 212
745 508
350 194
RNA/DNA
1.28 1.40
The amounts of DNA, RNA and protein were determined for the isolated nucleoli from 15-20 g liver as described in Materials and Methods. Each value presented was the average of two determinations.
Exptl Cell Res 71
72 T. Higasinakagawa et al. RNA synthetic activity RNA synthetic activity in terms of the incorporation of l*C-UMP into acid-insoluble material was determined for both nucleolar preparations and compared with each other. As clearly seen from fig. 11, the Mg2+-nucleoli exhibited a higher activity than the Ca2+-nucleoli at each time point studied. It may be said from this result that the Mg2+method is the method of choice when in vitro nucleolar RNA synthesis is studied. DISCUSSION
Fig. 10. Polyacrylamide gel electrophoresis patterns of acetic acid soluble proteins of the nucleoli. Proteins were extracted from isolated nucleoli with 67 % acetic acid and electrophoresed on a 15 % polyacrylamide gel as described in Materials and Methods. (a) Proteins of Mg2+-nucleoli; (b) split-gel comparison of the proteins of Mgz+-nucleoli (left) with those of Ca2+-nucleoli (right); (c) proteins of Ca2+-nucleoli.
Exptl Cell Res 71
Busch et al. [5] developed a procedure for isolation of nucleoli from rat liver employing magnesium acetate and spermine, by which satisfactory preparations were obtained. The present method utilizes only Mg2+-ions and the nucleoli prepared thereby are shown to possessmorphological and biochemical characteristics similar to those by Ca2+-method. Shanker Narayan & Birnstiel [16] isolated nucleoli from rat liver by sonicating the nuclei, which had been prepared with 5 mM MgCI,, in 0.34 M sucrose containing 0.5 mM MgCl, and 5-10 pg/ml polyvinylsulfate. They reported that the release of ribonucleoprotein particles was poor when nucleoli were isolated with Ca2+-method but that the Mg2+-nucleoli prepared by them contained more chromatin than those by Ca2+-method. The labeling profile (10 min with 14C-erotic acid) of the RNA extracted from their Mg2+-nucleoli were considerably contaminated with polydisperse RNA associated with chromatin fraction. Moreover, the appearance of the peak at about 20s region in their profile could possibly be the degradation products of rapidly labeled nucleolar 45s RNA molecule. In the present procedure, however, neither such contamination of the nucleoli by chromatin fraction nor degradation of RNAs was observed. The
Isolation of nucleoli
73
teins to form ribonucleoprotein structures of the nucleolus [14], the extraction and characterization of which have been performed by several workers [16-191 as well as in this laboratory (Higashinakagawa & Muramatsu. In preparation). Comparison of RNA synthetic activity of Ca2+- and Mg2+-nucleoli clearly shows that Mg2+-nucleoli possess a higher activity in vitro. The observed lower activity of the Ca2+-nucleoli may at least in part be due to the inhibitory effect of Ca2+-ions on the DNA-dependent RNA polymerase molecule [2]. It may be concluded from these results that the Mg2+-nucleoli are more suitable for the study of nucleolar ribonucleoprotein particles and the nucleolar RNA synthesis in 10 15 0+----vitro. The detailed study of RNA synthesis Fig. Il. Abscissa: min; ordinate: W-UMP incorpowith Mg2+-nucleoli and with RNA polyrated (cpm/mg DNA). O-O, Mg2+-nucleoli; O-O, Ca2+-nucleoli merase solubilized from Mg2+-nucleoli acComparisoh of in vitro RNA synthetic acitivy of cording to Roeder & Rutter [20] will be Mg2+-nucleoli with that of Caa+-nucleoli. The reaction mixture contained in a final volume of 1.Oml: published elsewhere (Higashinakagawa & 10 fig pyruvate kinase, 0.5 pmole each phosphoenol Muramatsu. In preparation). pyruvate, 1 pmole ATP, 0.125 pmole of GTP and CTP, 1.6 pmoles MnCl,, 25 pmoles Tris-HCl (pH Another advantage of the present method 7.9), 0.25 pmole dithiothreitol, 0.2 ,&i of W-UTP is to provide a better procedure for the pre(uridine+W-5’-triphosphate, 51 mCi/mmole, purchased from The Radiochemical Centre) and nucle- paration of nucleoli from regenerating rat oli from 1.5 g liver suspended in 0.34 M sucrose- 1 liver, in which nuclei as well as nucleoli are mM MgCl,. The reaction mixture was incubated for various periods at 37°C and then rapidly chilled in relatively fragile and disintegrated more an ice bath. 0.1 ml of 1 mg/ml UTPJ2C was added easily with the sonication procedure usually to each tube followed by 2 ml of 10 % trichloroacetic acid (TCA), 1 % NadP,O,. After 30 min at WC, the employed [21]. Mg2+-ions in the sonication resulting precipitate was transferred onto the glass fibre disc. and washed 6 times with 5 ml of 5 % medium are apparently effective in making TCA, 1 % Na4P20,, and finally once with 5 ml of these hypertrophied nucleoli more resistant ethanol. The discswere dried under an infrared lamp and counted in a liquid scintillation spectrometer to the sonication. using toluene scintillator. The activity was expressed The data presented above altogether sugin cpm/mg DNA. gest that the present method will serve as an alternative procedure for the isolation of fact that the RNA/DNA ratio was around nucleoli for the study of RNA and protein, 1.3 as well as the finding (in preparation) especially for the study of ribonucleoprotein that the RNA polymerase extracted from particles as well as enzymes of the nucleolus. these preparations was only peak I molecule (nucleolar RNA polymerase) of Roeder & The authors wish to thank Dr K. Ishikawa, Niigata Rutter [20]-all point to the purity of these University, for the useful information as to the splitgel electrophoresis and Dr T. Utakoji for taking phopreparations. The nucleolar RNA molecules tographs of the preparations under light microscope. are considered to be complexed with proThe excellent technical assistances of Messrs K.
I I,/
IJ II I’/
Exptl Cell Res 71
14
T. Higashinakagawa
et al.
Noguchi, T. Nashiro and M. Sakai are gratefully acknowledged. This work was supported in part by grants from the Ministry of Education of Japan.
REFERENCES 1. Muramatsu, M, Smetana, K & Busch, H, Cancer res 23 (1963) 510. 2. Weiss. S B. Proc natl acad sci US 46 (1960) 1020. 3. Dixon, M ‘& Webb, E C, The enzymes, p. 422. Academic Press, New York (1964). 4. Spirin, A S & Gavrilova, L P, The ribosomes, p. 63. Springer-Verlag, Berlin, Heidelberg and New York (1969). 5. Busch, H, Shanker Narayan, K & Hamilton, J, Exptl cell res 47 (1967) 329. 6. Spahr, P E, J mol biol 4 (1962) 395. 7. Ballard, P L & Williams-Ashman, H G, J biol them 241 (1966) 1602. 8. Burton, K, Biochem j 62 (1956) 315. 9. Fleck, A & Munro, H N, Biochim biophys acta 55 (1968) 571.
Exptl
Cell Res 71
10. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 11. Muramatsu, M, Shimada, N & Higashinakagawa, T, J mol biol 53 (1970) 91. 12. Leboy, P S, Cox, E C & Flaks, J G, Proc natl acad sci US 52 (1964) 1367. 13. Muramatsu, M, Higashinakagawa, T, Ono, T & Sugano, H, Cancer res 28 (1968) 1126. 14. Bernhard, W, Exptl cell res, suppl. 6 (1959) 17. 15. Muramatsu, M, Hodnett, J L, Steele, W J & Busch, H, Biochim biophys acta 123 (1966) 116. 16. Shanker Narayan, K & Birnstiel, M L, Biochim biophys acta 190 (1969) 470. 17. Warner, J R & Soeiro, R, Proc natl acad sci US 58 (1967) 1984. 18. Liau, M C & Perry, R P, J cell bio142 (1969) 272. 19. Izawa, M & Kawashima, K, Biochim biophys acta 174 (1969) 124. 20. Roeder, R G & Rutter, W J, Proc natl acad sci US 65 (1970) 675. 21. Muramatsu, M & Busch, H, J biol them 240 (1965) 3960. Received July 15, 1971 Revised version received November 25, 1971
ISOLATION OF NUCLEOLI FROM RAT LIVER IN THE PRESENCE OF MAGNESIUM IONS T. HIGASHINAKAGAWA’*,
M. MURAMATSU’**
and H. SUGAN02
Departments of ‘Chemistry and 2Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, Tokyo, Japan
SUMMARY A procedure for the isolation of nucleoli from rat liver in the presence of Mg2+ ions was developed. Addition of 0.05 mM MgC& to the 0.34 M sucrose in which isolated nuclei were suspended for sonication was found to protect nucleoli when nuclei prepared with 2.3 M sucrose containing 10 mM MgCl, were disrupted completely by sonication. The nucleolar preparation possesses almost identical properties with those of nucleoli prepared by the conventional Ca2+-method in the following points: (1) morphological characteristics as revealed by light and electron microscopy; (2) RNA/DNA ratio of around 1.3; (3) sedimentation profiles and labeling patterns of nucleolar RNA; (4) polyacrylamide gel electrophoresis of acetic acid soluble proteins. Furthermore, Mg2+-nucleoli showed a higher RNA synthetic activity than Ca2+-nucleoli. In addition, this method has proved to be advantageous in the case of regenerating liver in which nuclei and nucleoli are somewhat more fragile to sonication.
Isolation of nucleoli from rat liver cells has the aid of Mg2+ ions and spermine, by which been reported by Muramatsu et al. [l], almost identical nucleolar preparations were where 3.3 mM Ca2+ ions were added in the obtained with regard to morphological criinitial homogenization medium. In view of teria. The nucleolar RNA extracted from the inhibitory effects of Ca2+ ions and the these preparations was almost indistinguishstimulatory effects of Mg2+ ions on some able from that extracted from Ca2+-nucleoli. enzymatic activities [2, 31 and the ability of However, the addition of spermine may not Mg2+ ions to preserve the integrity of ribo- be suitable for detailed biochemical analyses nucleoprotein particles [4], it is desirable of various nucleolar components, such as to have an alternative procedure for the ribonucleoproteins and enzymes, because of isolation of nucleoli that utilizes Mg2+ ions the biological activities of polyamines [6, 71. rather than Ca2+ions. During the course of studies on the nuBusch et al. [5] developed a procedure for cleolar ribonucleoprotein particles, we have the isolation of nucleoli from rat liver with developed a procedure for the isolation of nucleoli with Mg2+ ions without utilizing * Present address: Mitsubishi-Kasei Institute of Life Sciences, c/o Central Research Laboratory, Mitother additives including spermine. This subishi Chemical Industries Limited, 290 Hisamotopaper describes the isolation procedure and kamoicho, Kawasaki, Japan, * * Present address: Department of Biochemistry, some properties of the isolated nucleoli preTokushima University School of Medicine, Tokupared in this manner. shima, Japan. 5-721803
Exptl Cell Res 71
66
T. Higashinakagawa et al.
MATERIALS AND METHODS Isolation of nuclei and nucleoli The procedure was essentially the same as that of Caz+-method [1] except that the CaZ+ ions were replaced with Mg2+ ions at a modified concentration. All operations were carried out at 2-4”C. The perfused livers were weighed, minced and homogenized with three up and down strokes in 10 vol of 2.3 M sucrose-10 mM MgCI, in a loosely fitting PotterElvehiem type homogenizer with a Teflon pestle. The homogenate was filtered through four layers of cheese cloth and centrifuged down at 40 000 g for 1 h. The nuclear pellet was suspended in 0.34 M sucrose -0.05 mM MgC12, gently homogenized and sonicated in 20 ml batches in a Kubota Ultra Sonic Generator, Model KMS-250 (10 kc/set) at an output of 200 W for 45 to 60 sec. Thesonicatewasrapidly checked for the extent of the disruption of nuclei under a light microscope. When virtually all the nuclei were destroyed, the sonicate was underlaved with 20 ml of 0.88 M sucrose-O.05mM M&l., and centrifuged at 2 000 g for 20 min in a refrigerated centrifuge. The supernatant was discarded and the inside of the centrifuge tube was wiped well with a sheet of clean tissue paper. The resulting pellet contained purified nucleoli. Isolation of nucleoli from regenerating rat liver could be performed in a similar manner, although a shorter sonication time, approx. 20 set, was found to be sufficient. In these procedures the concentration of MgCl, in the initial homogenizing medium (10 mM) and the sonication medium (0.05 mM) was found to be critical for the effective disruption of nuclei and for the maximal yield of nucleoli.
Determination of DNA, RNA and protein DNA was determined with Burton’s procedure [8], and RNA was determined by the method of Fleck & Munro [9]. Determination of protein was performed according to the method of Lowry et al. [lo] with bovine serum albumin as standard.
RNA extraction and sedimentation analysis RNA was extracted from the nucleolar pellet with SDS-hot phenol procedure and subjected to sedimentation analysis as described previously [ll].
Polyacrylamide gel electrophoresis of nucleolar acetic acid-soluble proteins Nucleolar preparations obtained with either Ca2+or MaZ+-method were suspended in 67 % acetic acid and ailowed to stand with occasional shaking in the cold overnight. The suspension was centrifuged at 20 000 g fo; 20 min at O’C and the supematant was dialysed against 6 M urea, 15 % sucrose. Fifty to 100 pg of proteins were electrophoresed on a 15 % polyacrylamide gel containing 6 M urea. Electrophoresis was conducted under a constant current of 1 mA/column for approx. 15 h in the cold using Exptl Cell Res 71
glycine-acetate buffer containing 6 M urea (pH 4.0). The gels were stained with 1 % amido-schwarz 1OB in 10 % acetic acid, destained by standing in 10% acetic acid. In order to compare the proteins of Ma2+-nucleoli with those of Ca2+-nucleoli. the splitgei technique [12] was employed. In this-procedure the two protein samples were electrophoresed in a single gel column and were compared in a more direct and rigorous manner.
Electron microscopy Electron microscopic observations of the isolated nuclear and nucleolar preparations were performed as described previously [13].
Assay of RNA synthetic activity The activity of the isolated nucleoli to incorporate [‘“Cl UMP into acid-insoluble fraction was measured as described previously [ll].
RESULTS Concentrations of magnesium chloride
In the present experiment, a variety of concentration of MgCl, in the initial hypertonic sucrosemedium and in the sonication medium was examined. In general, Mg2+ ions seemed to render the nuclei resistant to sonication. The combination of 2.3 M sucrose- 2 10 mM MgCl, and 0.34 M - 1 0.05 mM MgCl, gave the most satisfactory results. Addition of more than 0.1 mM MgCl, to the sonication medium made the nuclei too hard to be broken within 1 or 2 min, and the final nucleolar preparation was grossly contaminated with unbroken nuclei. On the other hand when no Mg2+ ions were included in the sonication medium, the nucleoli were not sufficiently resistant to the sonication resulting in a lower recovery. Nucleolar preparations from regenerating rat liver were obtained similarly but in this case sonication of only 20 set or so was found to be sufficient to destroy all the nuclei and release the nucleoli. Sonication for a longer period resulted in the disintegration of hypertrophied nucleoli of regenerating liver.
Tion of nucleoli
67
Fig. I. Rat liver nuclei isolated with 10 mM MgCl, in the homogenization medium, staLinedwith azure C. Fig. 2. Higher magnification of fig. 1.
Exptl Cell Res 71
68
T. Higashinakagawa et al.
Fig. 3. Rat liver nucleoli isolated from nuclei of fig. 1 by sonicating fhe.m in 0.34 M sucrose MgCl,, stained with azure C. The blurred spots are in fact nucleoh shghtly out of focus. Fig. 4. Higher magnification of fig. 4.
Exptl Cell Res 71
containing 0.05 mM
Isolation of nucleoli 69
Fig. 5. Nuclei isolated from the 18-h regenerating rat liver with 10 mM MgCI, in the hor nogenization nTedium, stained with Azure C. Fig. 6. Nucleoli isolated from nuclei of fig. 4 by sonicating them in 0.34 M sucrose contai ining 0.05 mM MgCL stained with Azure C. Some of the nucleoli are shown as blurred spots because they are out of focus.
Exptl Cell Res 71
70
T. Higashinakagawa et al.
Fig. 7. Electron micrograph of rat liver nuclei isolated with Mg2+-method. x 3 300. Fig. 8. Electron micrograph of the isolated nucleolus of rat liver. Note the granular ribonucAeoprotein strucl :ures within the nucleolus. x 40 000.
Exptl Cell Res 71
Isolation of nucleoli
71
Morphology of isolated nuclei and nucleoli
The purity of the nuclear and nucleolar preparations is shown in figs l-8. The nuclear preparation showslittle cytoplasmic contaminations (figs 1, 2, 5, 7) and retains nucleolar structure which is well stained with Azure C (figs 1, 2, 5). The nucleolar preparations contain very little unbroken nuclei nor aggregated chromatin (figs 3, 4, 6). The nucleolar fine structures are presented in fig. 8. As already known, the ribosome-like granular structures are seen [14]. Figs 5 and 6 represent the nuclear and nucleolar preparations isolated from 18 h-regenerating rat liver with the present procedure. The large size and somewhat irregular shape of the nucleoli characteristic of regenerating liver can be seen. Composition of isolated nucleoli
The amounts of DNA, RNA and protein were determined for the nucleoli isolated with Mg2+-procedure. The results are presented in table 1. The RNA/DNA ratio is around 1.3, which is a little higher than that of Ca2+-nucleoli (1.02) [I].
TOP
BOTTOM
Fig. 9. Abscissa: tube number; ordinate: (left) Azs4; (right) cpm. -, Azs4; 0 - 0, radioactivity. Sedimentation profile of RNA extracted from the nucleoli isolated with Mg2+-method. Rats were injected intravenously 50 ,uCi per rat of aH-orotic acid 10 min prior to sacrifice. Nucleoli were isolated with Mg2+-method as described in Materials and Methods. RNA was extracted from the nucleolar pellet with SDS-hot phenol procedure and placed on a lo-40 % (w/w) sucrose gradient buffered in 0.01 M Naacetate, 0.1 M NaCl, 1 mM EDTA (pH 5.1) and centrifuged in a SW 25.3 rotor of Spinco L2 Ultracentrifuge at 24 500 rpm for 20 h at 4°C. The gradient was fractionated with the aid of an ISCO gradient fractionater, Model D, with an automatic optical density recorder at 254 nm. Each fraction was assayed for radioactivity in a liquid scintillation spectrometer as described previously [12].
In fig. 9 is shown the sedimentation profile of RNA extracted from Mg2+-nucleoli. The optical density profile clearly shows the existence of main peaks of 45S, 35S, 28 S and
4-6s RNAs and some other minor peaks, quite similar to the profile of RNA from Ca2+-nucleoli. It is also clear from the labeling pattern that at 10 min labeling with 3Herotic acid, the radioactivity was distributed exclusively over 45s region, as was also the case with Ca2+-nucleoli [15]. These data clearly indicate the intactness of the RNA of the nucleoli prepared with Mg2+-method.
Table 1. Composition of the nucleoli isolated
Acetic acid-soluble proteins of the nucleoli
with Mg2f-method
Proteins were extracted from the isolated nucleoli with 67 % acetic acid and electrophoresed on the polyacrylamide gel. To compare the proteins of Mg2+-nucleoli with those of Ca2+-nucleoli, proteins were extracted from Ca2+-nucleoli and electrophoresed in the split-gel column. No appreciable difference was detected between the two protein samples as shown in fig. 10.
Integrity of nucleolar RNA
RNA
Protein
Ewt (A
DNA
old
ocd
21
448 212
745 508
350 194
RNA/DNA
1.28 1.40
The amounts of DNA, RNA and protein were determined for the isolated nucleoli from 15-20 g liver as described in Materials and Methods. Each value presented was the average of two determinations.
Exptl Cell Res 71
72 T. Higasinakagawa et al. RNA synthetic activity RNA synthetic activity in terms of the incorporation of l*C-UMP into acid-insoluble material was determined for both nucleolar preparations and compared with each other. As clearly seen from fig. 11, the Mg2+-nucleoli exhibited a higher activity than the Ca2+-nucleoli at each time point studied. It may be said from this result that the Mg2+method is the method of choice when in vitro nucleolar RNA synthesis is studied. DISCUSSION
Fig. 10. Polyacrylamide gel electrophoresis patterns of acetic acid soluble proteins of the nucleoli. Proteins were extracted from isolated nucleoli with 67 % acetic acid and electrophoresed on a 15 % polyacrylamide gel as described in Materials and Methods. (a) Proteins of Mg2+-nucleoli; (b) split-gel comparison of the proteins of Mgz+-nucleoli (left) with those of Ca2+-nucleoli (right); (c) proteins of Ca2+-nucleoli.
Exptl Cell Res 71
Busch et al. [5] developed a procedure for isolation of nucleoli from rat liver employing magnesium acetate and spermine, by which satisfactory preparations were obtained. The present method utilizes only Mg2+-ions and the nucleoli prepared thereby are shown to possessmorphological and biochemical characteristics similar to those by Ca2+-method. Shanker Narayan & Birnstiel [16] isolated nucleoli from rat liver by sonicating the nuclei, which had been prepared with 5 mM MgCI,, in 0.34 M sucrose containing 0.5 mM MgCl, and 5-10 pg/ml polyvinylsulfate. They reported that the release of ribonucleoprotein particles was poor when nucleoli were isolated with Ca2+-method but that the Mg2+-nucleoli prepared by them contained more chromatin than those by Ca2+-method. The labeling profile (10 min with 14C-erotic acid) of the RNA extracted from their Mg2+-nucleoli were considerably contaminated with polydisperse RNA associated with chromatin fraction. Moreover, the appearance of the peak at about 20s region in their profile could possibly be the degradation products of rapidly labeled nucleolar 45s RNA molecule. In the present procedure, however, neither such contamination of the nucleoli by chromatin fraction nor degradation of RNAs was observed. The
Isolation of nucleoli
73
teins to form ribonucleoprotein structures of the nucleolus [14], the extraction and characterization of which have been performed by several workers [16-191 as well as in this laboratory (Higashinakagawa & Muramatsu. In preparation). Comparison of RNA synthetic activity of Ca2+- and Mg2+-nucleoli clearly shows that Mg2+-nucleoli possess a higher activity in vitro. The observed lower activity of the Ca2+-nucleoli may at least in part be due to the inhibitory effect of Ca2+-ions on the DNA-dependent RNA polymerase molecule [2]. It may be concluded from these results that the Mg2+-nucleoli are more suitable for the study of nucleolar ribonucleoprotein particles and the nucleolar RNA synthesis in 10 15 0+----vitro. The detailed study of RNA synthesis Fig. Il. Abscissa: min; ordinate: W-UMP incorpowith Mg2+-nucleoli and with RNA polyrated (cpm/mg DNA). O-O, Mg2+-nucleoli; O-O, Ca2+-nucleoli merase solubilized from Mg2+-nucleoli acComparisoh of in vitro RNA synthetic acitivy of cording to Roeder & Rutter [20] will be Mg2+-nucleoli with that of Caa+-nucleoli. The reaction mixture contained in a final volume of 1.Oml: published elsewhere (Higashinakagawa & 10 fig pyruvate kinase, 0.5 pmole each phosphoenol Muramatsu. In preparation). pyruvate, 1 pmole ATP, 0.125 pmole of GTP and CTP, 1.6 pmoles MnCl,, 25 pmoles Tris-HCl (pH Another advantage of the present method 7.9), 0.25 pmole dithiothreitol, 0.2 ,&i of W-UTP is to provide a better procedure for the pre(uridine+W-5’-triphosphate, 51 mCi/mmole, purchased from The Radiochemical Centre) and nucle- paration of nucleoli from regenerating rat oli from 1.5 g liver suspended in 0.34 M sucrose- 1 liver, in which nuclei as well as nucleoli are mM MgCl,. The reaction mixture was incubated for various periods at 37°C and then rapidly chilled in relatively fragile and disintegrated more an ice bath. 0.1 ml of 1 mg/ml UTPJ2C was added easily with the sonication procedure usually to each tube followed by 2 ml of 10 % trichloroacetic acid (TCA), 1 % NadP,O,. After 30 min at WC, the employed [21]. Mg2+-ions in the sonication resulting precipitate was transferred onto the glass fibre disc. and washed 6 times with 5 ml of 5 % medium are apparently effective in making TCA, 1 % Na4P20,, and finally once with 5 ml of these hypertrophied nucleoli more resistant ethanol. The discswere dried under an infrared lamp and counted in a liquid scintillation spectrometer to the sonication. using toluene scintillator. The activity was expressed The data presented above altogether sugin cpm/mg DNA. gest that the present method will serve as an alternative procedure for the isolation of fact that the RNA/DNA ratio was around nucleoli for the study of RNA and protein, 1.3 as well as the finding (in preparation) especially for the study of ribonucleoprotein that the RNA polymerase extracted from particles as well as enzymes of the nucleolus. these preparations was only peak I molecule (nucleolar RNA polymerase) of Roeder & The authors wish to thank Dr K. Ishikawa, Niigata Rutter [20]-all point to the purity of these University, for the useful information as to the splitgel electrophoresis and Dr T. Utakoji for taking phopreparations. The nucleolar RNA molecules tographs of the preparations under light microscope. are considered to be complexed with proThe excellent technical assistances of Messrs K.
I I,/
IJ II I’/
Exptl Cell Res 71
14
T. Higashinakagawa
et al.
Noguchi, T. Nashiro and M. Sakai are gratefully acknowledged. This work was supported in part by grants from the Ministry of Education of Japan.
REFERENCES 1. Muramatsu, M, Smetana, K & Busch, H, Cancer res 23 (1963) 510. 2. Weiss. S B. Proc natl acad sci US 46 (1960) 1020. 3. Dixon, M ‘& Webb, E C, The enzymes, p. 422. Academic Press, New York (1964). 4. Spirin, A S & Gavrilova, L P, The ribosomes, p. 63. Springer-Verlag, Berlin, Heidelberg and New York (1969). 5. Busch, H, Shanker Narayan, K & Hamilton, J, Exptl cell res 47 (1967) 329. 6. Spahr, P E, J mol biol 4 (1962) 395. 7. Ballard, P L & Williams-Ashman, H G, J biol them 241 (1966) 1602. 8. Burton, K, Biochem j 62 (1956) 315. 9. Fleck, A & Munro, H N, Biochim biophys acta 55 (1968) 571.
Exptl
Cell Res 71
10. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 11. Muramatsu, M, Shimada, N & Higashinakagawa, T, J mol biol 53 (1970) 91. 12. Leboy, P S, Cox, E C & Flaks, J G, Proc natl acad sci US 52 (1964) 1367. 13. Muramatsu, M, Higashinakagawa, T, Ono, T & Sugano, H, Cancer res 28 (1968) 1126. 14. Bernhard, W, Exptl cell res, suppl. 6 (1959) 17. 15. Muramatsu, M, Hodnett, J L, Steele, W J & Busch, H, Biochim biophys acta 123 (1966) 116. 16. Shanker Narayan, K & Birnstiel, M L, Biochim biophys acta 190 (1969) 470. 17. Warner, J R & Soeiro, R, Proc natl acad sci US 58 (1967) 1984. 18. Liau, M C & Perry, R P, J cell bio142 (1969) 272. 19. Izawa, M & Kawashima, K, Biochim biophys acta 174 (1969) 124. 20. Roeder, R G & Rutter, W J, Proc natl acad sci US 65 (1970) 675. 21. Muramatsu, M & Busch, H, J biol them 240 (1965) 3960. Received July 15, 1971 Revised version received November 25, 1971