John H. Frenster, Vincent G. Allfrey, and Alfred E. Mirsky

The Rockefeller Institute, New York, NY 10021

Extraction of Nuclei:
Centrifugation of Extracts
Intranuclear Metabolism of Particles:
Electron Microscopy of Particles:
Nuclear Origin of Particles:
Metabolism of Particles in a Nuclear-Free System:

It is also shown here that after extraction from the cell nucleus, the isolated nuclear ribonucleoprotein particles remain capable of active protein and RNA metabolism in a nucleus-free system.

Previous studies of the cell nucleus by electron microscopy have demonstrated the occurrence of discrete particles of 100-300 A. diameter in the nucleolus (5-7), on the lateral loops of lampbrush chromosomes (8), on the chromosomal rings of Balbiani (9), in the blebs (10) and pore annuli (11, 12) of the nuclear membrane, and throughout the nuclear sap (13). Particles of similar size are shown here to be extracted from the nuclei of calf thymus lymphocytes, and to be composed of sub-units of 15-25 A. diameter.

The heterogeneity of the RNA of the cell nucleus has been shown previously by radioautography of labelled nuclei (14), by isolation of labelled nuclear RNA species (15, 16), by detemination of the base composition of isolated nuclear RNA species (17), and by histochemical staining of nuclear ribonucleoprotein species throughout the mitotic cycle (18). Composition and metabolic heterogeneity is also found here in isolated nuclear ribonucleoprotein particles and presumably reflects distinct origins and functions for the different particles.

MATERIALS AND METHODS

Isolation and incubation of nuclei:

All procedures other than incubation were performed at 2^o C. Nuclei from calf thymus lymphocytes were isolated in 0.25 M sucrose - 0.003 M CaCl₂ (1) and were incubated at 37^o C in the presence of such C¹⁴-labelled precursors as DL-alanine-1-C¹⁴ , DL-lysine-1-C¹⁴, DL-leucine-1-C¹⁴, orotic acid-6-C¹⁴, or adenosine -8-C¹⁴ in the buffered sucrose-glucose medium supplemented with sodium (1) as described in detail in the legends to the figures.

Purification and extraction of nuclei:

The uptake of C¹⁴-precursors was stopped by rapid chilling, the inubation medium containing cytoplasmic fragments removed by centrifugation, the nuclei washed once with cold incubation medium, and the nuclei then succesively extracted three times by blending 1 min at 7,500 rpm with 5 volumes of cold 0.1 M tris (hydroxymethyl) aminomethane chloride (Tris Cl) buffer, pH 7.1. The resulting three Tris extracts were each separated from the nuclei by centrifugation at 1,000 g for 10 min. The extracts were then each purified by centrifugation at 3,000 g for 30 min to remove contaminating whole nuclei and particles greater than 7000 S.

Differential ultracentrifugation of extracts:

Each of the three Tris extracts was subjected to successive differential ultracentrifgation in the Spinco Model L Ultracentrifuge, No. 40 head, at 2^oC. Pellets were collected after successive centrifugations at 10,000 g_av, 33,000 g_av, and 105,000 g_av for 30 min, and at 105,000 g_av for 5 hr and 16 hr. The pellets formed were then washed twice with cold 0.1 M Tris (Cl), pH 7.1. Ranges of sedimentation constants for the particles in the pellets were estimated from the performance characteristics of the Spinco Model L No. 40 Rotor.

Measurement of protein, RNA, DNA, and radioactivity:

Cold trichloracetic acid was added to the pellets and supernates to final concentration of 5 percent trichloroacetic acid (TCA). Radioactivity in protein or RNA was measured as previously described (15) in a thin-window gas-flow counter, and the counts were corrected for self-absorption (19). Deoxyribonucleic acid (DNA) was determined by the diphenylamine reaction (20), and RNA by the p-bromphenylhydrazine reaction (21) on TCA hydrolystes obtained after heating for 30 min at 90^o C. Protein was determined as previously described (1).

Electron microscopy:

Electron micrographs of the ultracentrifuge pellets were obtained after fixation of the pellets in Veronal-buffered osmiun tetroxide pH 7.3, dehydration in alcohol, embedding in n-butylmethyl methacrylate, and sectioning on a Porter-Blum microtome. A Phillips Model EM-100 A electron microscope was used with intermediate lens and Kodak D-426 film yielding microscopic magnifications to 20,000 X. Subsequent magnifications to 240,000 X were obtained photographically.

Studies on a nucleus-free system:

For studies of leucine-1-C¹⁴ incorporation into proteins of isolated nuclear ribonucleoprotein particles in a nucleus-free system, the nuclei were isolated in 0.25 M sucrose-0.003 M MgCl₂, and freed of contaminating cytoplasmic fragments by two washings in an incubation medium of the following composition: 0.1875 M sucrose, 0.02 M glucose, 0.025 M Tris chloride, 0.0285 M NaCl, 0.008 M MgCl₂, pH 7.4. The washed nuclei were extracted with 0.1 M Tris Cl-0.003 M MgCl₂, pH 7.1. This 0.1 M Tris T extract was centrifuged at 3,000 g_av for 30 min, and the resulting nucleus-free supernate was centrifuged at 105,000 g_av for 60 min, yielding sedimented particles of 120-7000 S. The resulting supernate was brought to pH 5.2 with 1.0 M acetic acid, and the precipitate collected as nuclear pH 5 enzymes.

RESULTS AND DISCUSSION

Extraction of Nuclei:

Since 0.1 M Tris (hydroxymethyl) amininomethane buffer, pH 7.1 was a more efficient extractant of both proteins and RNA from the nuclei (Table 1) than the neutral 0.1 M potassium phosphate buffer used previously (1, 15, 17), Tris was used as the extracting buffer in all subsequent experiments.
 
 

TABLE 1. Comparison of Neutral Buffer Extractions of Nuclei.

Composition:	DNA mgm	Protein mgm	RNA mgm
Whole Nuclei-before extraction	1576	4785	105.5
pH 7.1 Buffer extracts: *
0.1 M Sodium phosphate	1.5	680	42.8
0.1 M Potassium phosphate	1.3	850	54.4
0.1 M Tris chloride	1.6	885	61.8
0.1 M Tris Cl-0.003 M CaCl2	0.2	720	50.1
0.1 M Tris Cl-0.003 M MgCl2	0.4	590	41.1

* Unsedimented after centrifugation at 3,000 g_av for thirty min.

Addition of either Ca⁺⁺ or Mg⁺⁺ did not enhance extraction. Up to 20 percent of proteins and 60 percent of RNA could be extracted from the nuclei with only minimal extraction of DNA, the particular yield varying with different preparations.

The yield of both proteins and RNA which were extracted declined from the first extract to the third (Table 2),
 
 

TABLE 2. Extraction by Neutral 0.1 M Tris Buffer of Protein and RNA from Nuclei Following Incubation with Radioactive Precursors.

Composition and  incorporation:			Alanine-1-C14 incorporation4			Adenosine-8-C14 incorporation5
	DNA mgm	Protein mgm	Counts cpm	Spec. Act. cpm/mgm	RNA mgm	Counts cpm	Spec. Act. cpm/mgm
Whole Nuclei1	1,440	4,220	625,000	148	52.2	131,800	2,520
>7000 S Particles2	3.2	...	...	...	5.38	1,380	256
<7000 S Particles3
Extract I	1.94	314	179,000	572	7.09	1,290	182
Extract II	1.52	172	98,200	571	3.20	2,680	837
Extract III	1.20	59	33,600	569	1.81	7,390	4,070
Residual Nuclei	1,428	3,360	218,000	65	33.1	97,200	2,960
Recovery, per cent	99.6	92.5	84.5	...	96.8	83.5	...

Incubation for sixty min. at 37^o C with agitation. Approximately 40 mgm (dry weight) of isolated nuclei per ml of incubation medium. Incubation medium of final concentration: 0.1875 M Sucrose, 0.02 M glucose, 0.0285 M NaCl, 0.025 M sodium phosphate, 0.0065 M MgCl₂, 0.0015 M CaCl₂, pH 6.8
¹After removal of cytoplasmic fragments soluble in incubation medium.
²Sedimented after centrifugation of extracts at 3,000 g_av for thirty min.
³Unsedimented after centrifugation of extracts at 3,000 g_av for thirty min.
⁴DL-alanine-1-C¹⁴, 0.56 uc/0.025 mgm added to each ml of alanine incubation medium.
⁵Adenosine-8-C¹⁴, 0.16 uc/0.239 mgm added to each ml of adenosine incubation medium.

but, while the specific activity of incorporated radioactivity was almost uniform in the proteins of all three extracts, the specific activity of the RNA of the third extract was almost 30 times that of the first extract (Table 2), suggesting that the third extract contained RNA which had been extracted from the nucleolus (15).

The time course of alanine-1-C¹⁴ incorporation into the proteins of the nuclei (Fig 1) shows a lag period of 10 to 20 min (1), followed by a progressive increase in labelling. The material which sedimented after centrifugation at 3,000 g for 30 min (>7000 S) resembled the protein of whole nuclei in its specific activity and was discarded. The incubation medium contained cytoplasmic fragments which could be identified by their appearence by electron microscopy; these showed insignificant incorporated radioactivity which was resistant to DNAase preincubation. The proteins of the third extract were always slightly less active than those of the first two extracts (Table 2, Fig. 1).

Fig. 1. Time course of incorporation of of alanine-1-C¹⁴ into proteins of of nuclei. Incubation conditions, media composition, and amount of isotope added same as in Table 2.

Centrifugation of Extracts:

When each of the three Tris extracts of nuclei which have been incubated with alanine-1-C¹⁴ is ultracentrifuged, an appreciable portion of the extracted proteins and their incorporated radioactivity can be sedimented as ribonucleoprotein pellets (Table 3).
 
 

TABLE 3. Nuclear Particle Isolation by Various Types of Centrifugation after Incubation of Nuclei with Alanine-1-C¹⁴

Protein Content and Specific Activity of  Centrifuge Pellet
Tris Extract No.	I	I	II	II	III	III
	mgm	cpm/mg	mgm	cpm/mg	mgm	cpm/mg
Method of centrifugation
Fixed angle uniform density1	55.4	398	49.2	442	9.8	374
Fixed angle gradient density2	30.2	227	16.6	307	5.3	305
Swinging bucket gradient density3	28.8	178	...	...	...	...

Incubation conditions, media composition, amount of isotope added, and 0.1 M Tris extraction same as Table 2.
¹Entire centrifuge tube filled uniformly with 0.1 M Tris extract of nucleus. Centrifugation in Spinco Model No. 40 Head for five hr at 105,000 g_av.
²Two volumes of 0.1 M Tris extract layered over one volume of 1.0 M sucrose-0.003 M MgCl₂ in each centrifuge tube. Centrifuged in Spinco Model L, No. 40 Head for five hr at 105,000 g_av.
³One volume of 0.1 M Tris extract layered over one volume of 1.0 M sucrose-0.003 M MgCl₂ in each centrifuge tube. 

Centrifugation in a fixed-angle head is the most convenient method and results in the highest yield of sedimented proteins. As an alternative, centrifugation across a density gradient separates the sedimentable ribonucleoprotein particles away from the less sedimentable soluble proteins in the extracts with which they are present. The particles so separated retain their incorporated radioactivity (Table 3).

Fig. 2. Differential ultracentrifugation of nuclear ribonucleoprotein particles. Nuclei extracted successively three times with five volumes of cold 0.1 M Tris(hydroxymethyl)aminomethane buffer pH 7.1. Each of the three Tris extracts subjected to differential ultracentrifugation as in schedule described in Methods and Materials.

When each of the three Tris extracts are subjected to differential ultracentrifugation (Fig. 2), discrete ranges of ribonucleoprotein particles are sedimented, each with its characteristic composition of protein and RNA. Protein/RNA ratios of the pellets ranged from 2 to 100. The 150-700 S pellet in each extract is especially interesting because of its protein/RNA ratio of 2/1. Nearly all of the extracted RNA could be sedimented (>6 S), but a considerable portion of the extracted proteins could not (<6 S).

Intranuclear Metabolism of Particles:

The different nuclear ribonucleoprotein particles show marked differences in their rate of incorporation of alanine-1-C¹⁴ into protein while in the nucleus (Fig. 3).

Fig. 3. Time course of incorporation of alanine-1-C¹⁴ into proteins of nuclear ribonucleoprotein particles. Incubation conditions, media composition, and amount of isotope added same as in Table 2. Differential ultracentrifugation of 0.1 M Tris extract in schedule described in Materials and Methods. 

No obvious precursor-product relationships between particles were evident in these simple time-course studies. Because of the known intimacy of the metabolism of protein and RNA in protein synthetic processes in the cytoplasm (3, 22), it was of interest to study the simultaneous intranuclear metabolism of protein and of RNA in the nuclear ribonucleoprotein particles of one individual. In such a study (Fig. 4),

Fig. 4. Concurrent incorporation of radioactive precursors into proteins and RNA of nuclear particles. Incubation conditions and media composition same as in Table 2. DL-lysine-1-C¹⁴,0.09 uc/0.05 mgm added to each ml of lysine incubation medium. Orotic acid-6-C¹⁴, 0.219 uc/0.02 mgm added to each ml of orotic acid incubation medium. Differential ultracentrifugation of 0.1 M Tris extracts I, II, and III as in schedule described in Materials and Methods.

the rates of protein and of RNA metabolism in a given particle range were parallel, being high for both protein and RNA in particles 700-2000 S and particles 6-20 S, and low for both protein and RNA in particles 2000-7000 S, 150-700 S, and 20-150 S.

Both the small amount of supernatant RNA (<6 S) and the larger amount of supernatant protein (<6 S) displayed very active intranuclear metabolism within each extract (Fig. 4). The significance of such active metabolism in these compounds is unknown. Preliminary observations suggest that both the carrier RNA and the pH 5.0 enzymes involved in amino acid activation and transfer in the nucleus (23) are sedimentable within 16 hr at 105,000 g_av(24), and thus of sedimentation constant greater than 6 S.

Electron Microscopy of Particles:

Electron micrographs of intact isolated nuclei before extraction with neutral buffer reveal the nuclei to be largely free of adherent cytoplasm, only 5 percent of isolated nuclei showing adherent cytoplasmic fragments. In favorable sections, the nucleolar region of the nucleus can be seen (Fig. 5a). Higher magnifications of such intact nuclei reveals the presence of large numbers of granules in both the nucleolus (Fig. 5b) and in the extra-nucleolar regions (Fig. 5c) of the nucleus. The granules are of two basic types, small solidly dense particles of 15 A. diameter, and slightly larger hollow particles with a dense periphery and a diameter of 25 A. Both types of particles are found arranged singly, in regular rings of 75-100 A. total diameter, or in irregular clusters of 75 A. diameter. After extraction with neutral buffers, there is an obvious change in the fine structure of the nuclei (Fig. 6), with a decrease in the number of granules, and a coarsening of the remaining nuclear structure.

Electron micrographs of isolated ribonucleoprotein particles after extraction from the nuclei by neutral buffer reveals the presence of large numbers of dense granules of 100 A. diameter (Fig. 7a). Higher magnifications of such granules (Fig. 7b) reveals them to be composed of rings and clusters of either hollow particles of 25 A. diameter each. Similar rings (Fig. 8a) and clusters (Fig. 8b) of 25 A. diameter solid particles are found in the ribonucleoprotein pellets of both the first and third Tris extracts.

Hollow particles with dense peripheries and a total diameter of 25 A. have been illustrated in the higher magnifications of electron micrographs of elementary chromosomal fibrils (Fig. 9 of ref. 25), of rings of nucleolar particles (Fig. 3 of ref. 7), of nuclear pore annuli granules (Fig. 7 of ref. 12), and of cytoplasmic microsomal granules (Fig. 7 of ref. 12). The possible relationship of such illustrated hollow particles to the ribonucleoprotein particles isolated here is of great interest but is, as yet, undetermined.

Nuclear Origin of Particles:

That the ribonucleoprotein particles are of nuclear and not of cytoplasmic origin can be shown in several ways.
First, it can be shown that in a sodium-supplented medium as here employed to label the proteins of the nuclei, virtually no labelling of the proteins of the cytoplasmic fragments (Fig. 1, Table 4) occurs, whereas the labelling of the proteins of the nucleus and its particles is brisk.
 
 

TABLE 4. Rates of Precursor Incorporation into Protein and RNA of Nuclear Fractions and Cytoplasm.

Precursor Incorporation:
	Into Protein	Into Protein	Into RNA	Into RNA
	lysine-1-C14 cpm/mgm 3	alanine-1C14 cpm/mgm 4	orotic acid-6-C14 cpm/mgm 5	adenosine-8-C14 cpm/mgm 6
Whole nucleus	13	148	5705	2520
Cytoplasmic fragments1	1	5	750	244
0.1 M Tris Extract I2	44	572	269	182
0.1 M Tris Extract II2	43	571	1215	837
0.1 M Tris Extract III2	35	569	7570	4070
Residual nucleus	6	65	929	2960

Incubation conditions and media composition same as in Table 2.
¹Fraction soluble in incubation medium.
²Supernatant after centrifugation at 3,000 g_av for thirty min.
³DL-lysine-1-C¹⁴, 0.09 uc/0.05 mgm added to each ml of lysine incubation medium.
⁴DL-alanine-1-C¹⁴, 0.56 uc/0.025 mgm added to each ml of alanine incubation medium.
⁵Orotic acid-6-C¹⁴, 0.219 uc/mgm added to each ml of orotic acid incubation medium.
⁶Adenosine-8-C¹⁴, 0.16 uc/0.239 mgm added to each ml of adenosine incubation medium.

Second, if the nuclei are pre-incubated with DNAase or with RNAase before incubation with alanine-1-C¹⁴ (Fig. 9),

Fig. 9. Effect of pre-incubation with nucleases on subsequent incorporation of alanine-1-C¹⁴ into proteins of nuclear particles. Fifteen min pre-incubation of whole nuclei at 37^o C in media as described in Table 2, with addition of crystalline pancreatic DNAase 0.25 mgm/ml or of RNAase 0.5 mg/ml, or of neither. 67.5 percent of DNA removed from whole nuclei by DNAase pre-incubation, and 26.2 percent of RNA removed from whole nuclei by RNAase pre-incubation. Subsequent incubation with DL-alanine-1-C¹⁴ as described in Table 2. Differential ultracentrifugation of Tris exracts I as in schedule described in Materials and Methods.

the rate of incorporation into the proteins of the nuclear particles is found to be sensitive to DNAase preincubation but not to RNAase preincubation. This is in marked contrast to the behavior of cytoplasmic particles, whose protein metabolism is exquisitely sensitive to RNAase preincubation (3, 4).

Third, when the nuclei are incubated in a potassium environment rather than in the usual (sodium) environment (Fig. 10),

Fig. 10. Effect of monovalent cation on incorporation of alanine-1-C¹⁴ into proteins of nuclear particles. Incubation conditions, isotope added, and media same as in Table 2 except in the potassium environment incubation, equimolar amounts of KCl, and potassium phosphate pH 6.8 were substituted for usual NaCl and sodium phosphate pH 6.8. Differential ultracentrifugation of Tris extracts I as in schedule described in Materials and Methods.

incorporation of alanine-1-C¹⁴ into the proteins of the particles is greatly decreased. This is in marked contrast to the behavior of cytoplasmic ribonucleoprotein particles, whose protein metabolism is increased by potassium and decreased by sodium (2).

Fourth, the particles obtained in the first Tris extract are composed of protein of high specific activity and RNA of low specific activity. The specific activity of this RNA is, in fact, lower than that in the cytoplasmic fragments contained in the incubation medium (fraction) (Table 4), suggesting that the RNA of the incubation medium cannot be contributing by contamination to the very low specific activity of the RNA of the first Tris extract.

Finally, the electron micrography described above reveals that the solid and hollow particles of 15-25 A. diameter extracted by neutral buffer from the nuclei resemble both in size and arrangement the particles seen in large numbers in the intact nuclei before extraction and in decreased numbers in the nuclei after extraction.

Metabolism of Particles in a Nucleus-Free System:

When isolated nuclear ribonucleoprotein particles are incubated in a nuclear-free system, they are found to be capable of active incorporation of leucine-1-C¹⁴ into the proteins of the isolated particles (Fig. 11). 

Fig. 11. Incorporation of Leucine-1-C¹⁴ into proteins of isolated nuclear ribonucleoprotein particles in a nuclear-free system. Isolation of nuclei, nuclear ribonucleoprotein particles 120-7,000 S, and nuclear pH 5 enzymes <120 S as described in Materials and Methods for studies on a nucleus-free system. The complete system consisted of: nuclear ribonucleoprotein particles 120-7,000 S (5.5 mgm protein, 1.23 mgm RNA, and <0.04 mgm DNA), nuclear pH5 enzymes < 120 S (0.96 mgm protein, 0.04 mgm RNA, and <0.001 mgm DNA), 0.20 mgm pyruvate kinase, 20 umoles sodium phosphoenolpyruvate (PEP), 2.0 umoles sodium ATP, 1.0 umole of sodium GTP and of sodium CTP, and L-leucine-1-C¹⁴ (2.0 uc/0.05 mgm) in a final volume of 2.0 ml of an incubation medium of following composition: 0.1875 M sucrose, 0.02 M glucose, 0.025 M TrisCl, 0.0285 M NaCl, 0.008 M MgCl₂, pH 7.4. Incubated at 37^o C in Air. 

A study of the requirements for such incorporation in a nucleus-free system (Table 5),
 
 

TABLE 5. Requirements for Incorporation of Leucine-1-C¹⁴ into Proteins of Isolated Nuclear Ribonucleoprotein Particles in a Nuclear-Free System¹.

			cpm/mgm Protein
Complete system			62
"	minus	Nuclear Particles	9
"	"	Nuclear pH 5 Enzymes	33
"	"	PEP	20
"	"	ATP	27
"	"	GTP	26
"	"	CTP	68

¹Isolation of nuclei, nuclear ribonucleoprotein particles 120-7000 S, and nuclear pH enzymes <120 S as described in Materials and Methods: Studies on a nuclear-free system. Incubation medium, isotope, and additions as in Figure 11 (above), except for deletions as indicated. Incubated 37^oC 60 min in air.

shows the process to require the nuclear pH 5 enzymes, adenosine triphosphate (ATP), guanosine triphosphate (GTP), and a source of generating ATP. Cytidine triphosphate (CTP) was slightly inhibitory to the process. The system contained only trace amounts of measureable DNA.

SUMMARY

Ribonucleoprotein particles can be extracted by neutral buffer solutions from isolated nuclei of calf thymus lymphocytes. Such particles can be fractionated and characterized by their ease of extraction, their sedimentability, their protein and RNA composition, and by their rate of protein and RNA metabolism in the nucleus. The rates of protein and RNA metabolism in the nucleus are parallel in each class of particles. The particles are revealed by electron micrography to consist of elementary units of 15-25 A. diameter, arranged in rings or clusters. Metabolic evidence suggests that certain of the classes of isolated particles may be derived from the nucleolus of the nucleus. Isolated particles extracted from the cell nucleus remain capable of incorporating precursors into their constituent proteins in a nucleus-free system.

The able technical assistance of Mr. Don Jasper in the preparation of the electron micrographs was appreciated.
A partial account has been presented in: Blood 14, 1258 (1959).

This research has been supported in part by Public Health Service Grant 4919. J.H.F. is assisted by a post-doctoral fellowship grant (PF-30) from the American Cancer Society.

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1. Frenster JH, Allfrey VG, and Mirsky AE, "In-Vitro Incorporation of Amino Acids into the Proteins of Isolated Nuclear Ribosomes", Biochim. Biophys. Acta 47: 130-47 (1961).

2. Frenster JH, "DNA as a Direct Template for Protein Synthesis", Editor's Commentary, January 1, 2004.