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An Analysis of the Rapidly Synthesized Ribonucleic Acid of the Normal Human Lymphocyte by Agarose-Polyacrylamide Gel Electrophoresis" Paul E. Neimant and Patrick H. Henry
ABSTRACT: Previous studies have demonstrated that the normal human lymphocyte in vitro rapidly incorporates labeled precursors into large molecular weight (30-100 s) non-rRNA which is heterodisperse in sucrose density gradient analysis. The development of mixed agarose-polyacrylamide gels for electrophoresis of RNA allowed a more detailed study of this material. Utilizing gel columns composed of 1 . 7 6 x acrylamide, 0.2% bisacrylamide, and 0.25 agarose, a pore size is achieved which will readily admit and fractionate the large molecular weight RNAs synthesized by lymphocytes. Discrete peaks appear after about 1 hr of in vitro labeling, and by 6 hr, fractions of nominal molecular weights corresponding to 90, 75, 67, 50, and 40 S are consistently resolved. Cell fractionation and pulse-chase studies indicate that these molecules are turning over in the nucleus and do not bear a clear
T
he nucleus of eucaryotic cells contains a heterogeneous class of large molecular weight RNA which undergoes rapid synthesis and degradation but whose function remains obscure. Its presence has been confirmed in many different cell types in several different laboratories (Scherrer et al., 1966; Attardi et al., 1966; Houssais and Attardi, 1966; Warner et al., 1966; Penman et al., 1966). Several properties have been described which characterize this material including heterodisperse (10-90 S) distribution on sucrose gradients, DNA-like (G C) content, nucleoplasmic origin (Soeiro et a[., 1966; Scherrer et al., 1966), and high efficiency of hybrid formation with homologous DNA (Perry et nl., 1964; Birnboim et al., 1967). Despite these characteristics, several studies indicate that this material turns over in the nucleus, and that there is no clear precursor relationship to cytoplasmic polyribosome-bound mRNA (Attardi et al., 1966; Soeiro et al., 1968; Penman et al., 1968). The experiments reported here were performed with small lymphocytes isolated from the blood of normal human subjects in short-term tissue culture. These small cells, composed of a compact nucleus and a thin rim of cytoplasm, are nondividing and do not synthesize DNA under standard conditions. Studies previously reported from this laboratory (Torelli et al., 1968) demonstrated that the vast majority of the nucleic acids synthesized by these cells in short-term cultures were large molecular weight rapidly labeled heterogeneous RNA species with properties on sucrose gradient analysis which were in-
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* From the Department of Medicine, University of Washington, Seattle, Washington, and the Department of Medicine, University of Missouri, Columbia, Missouri. Receiced October 12, 1970. This project was supported in part by Award CA-AM-11438 from the National Institutes of Health. Paul Neiman is a special research fellow of the National Cancer Institute (l-F3-CA-40,487).
relationship to cytoplasmic heterodisperse RNA which requires much longer labeling periods to detect. Base ratio analysis of individual peaks demonstrates a high U content and (G C) contents ranging from 36 to 50 %. RNA extracted from different regions of the gel formed hybrids with homologous DNA to varying degrees. The greatest degree of hybrid formation occurred with RNA molecules larger than those in the discrete peaks. They were distributed in a heterodisperse fashion on the gel with molecular weights as high as 250 S. Stimulation of lymphocytes with phytohemagglutinin did noi produce major qualitative changes in the monodisperse RNA peaks. The acrylamide gel technique has thus resolved several distinct molecular species of rapidly labeled nuclear RNA which were synthesized along with the more heterodisperse species and which could not be identified with other methods.
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distinguishable from those described above for other cell types. Synthesis of rRNA was shown to be minimal in these cells, and therefore does not complicate studies of rapidly synthesized RNA. The development by Peacock and Dingman (1968) of a series of mixed agarose-polyacrylamide gels with pore sizes large enough to admit and fractionate RNA molecules of molecular weight as high as 108 made available a method of fractionating populations of RNA molecules more completely than was possible with previously available techniques. In the hope of better characterizing lymphocyte heterodisperse nuclear RNA, we have applied a technique utilizing mixed acrylamide-agarose gel columns to the analysis of rapidly labeled RNA from normal human lymphocytes. The results of this study indicate that several apparently discrete, and heretofore undescribed, molecular species of low (G C) content can be resolved by this technique and demonstrated to be turning over within the nucleus. These species of RNA are synthesized on a background of more heterodisperse RNA molecules which form hybrids with DNA with higher efficiencies than do the monodisperse molecular species.
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Experimental Section Materials
Nylon fiber (Leuko-Pak) was purchased from Fenwal Corp., Morton Grove, Ill., and thoroughly washed in distilled water to remove toxic materials. [5-aH]Uridine (26 c i / mmole), NCS solubilizer, and toluene-phosphor (Liquifluor) were obtained from Amersham-Searle Corp. [a2P]Phosphoric acid, carrier free, is a product of the New England Nuclear Corp. The water-jacketed disc gel apparatus was purchased from Buchler Instruments and thermally regulated by means of a refrigerated water bath with a n external circulating pump. B I O C H E M I S T R Y , VOL.
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Acrylamide monomer and bis were purchased from Kodak Corp. and recrystalized. TEMED, also a Kodak product, was redistilled. Agarose was "Sedkem" distributed by Baush & Lomb. Cacodylic acid was purchased from Fisher Chemicals and recrystalized. The apparatus for horizontal slicing of the gel columns into 1.24-mm disks was made by Earl Sandbeck of Scientific Instruments, Baltimore, Md. Prepared thin-layer foils of MN-cellulose 300 were purchased from Brinkman Instrument Corp. Photoflow is a Kodak product. Phytohemagglutinin was Difco, PHA-P.
AND H E N R Y
0.08 M sucrose-0.0033 M CaCI,, pH 7.4), 1 m1/108 cells, in a Dounce homogenizer and subjected to IO strokes of a tightfitting "B" pestle. The homogenate was made 0.1 % with respect to Triton X-100 and 10 more strokes were applied. Following centrifugation at 200g for 5 min, the supernatant fraction was aspirated and extracted for cytoplasmic RNA in the usual way. The nuclear pellet was washed once in 0.15 M NaCl-O.01 M Tris-0.0033 M CaCh (pH 7.4) and the nuclei lysed by suspension in hypertonic buffer (1.5 M NaC1-0.05 M MgClZ-0.01 M Tris, p H 7.4). The gelatinous mass of DNA thus released was degraded for 2 min at room temperature by Methods addition of 40 pg of electrophoretically purified deoxyriboPreparation and Culture of' Lymphocytes. Leukocyte-rich nuclease per lo8 nuclei. The usual RNA extraction procedure plasma was obtained by the sedimentation, at 37", of hepawas then carried out. By phase microscopy, this combination rinized fresh whole blood drawn from normal human subjects of cytoplasmic swelling and detergent treatment effectively with 0.1 volume of 6 % Dextran (mol wt 75,000) in normal strips the thin rim of cytoplasm of these small cells. This was saline. Polymorphonuclear leukocytes and monocytes were confirmed by electron microscopic study (P. E. Neiman and removed from the plasma by passage through a nylon fiber P. H. Henry, unpublished observations) which also revealed column according to a modification of the method of Greenthe loss of the outer nuclear membrane. The retention in the Walt et al. (1962; Torelli et al., 1968). The cells, 99% small nuclear fraction of 90-95Z of the cellular DNA and the same percentage of radioactive RNA following a 15-min lymphocytes, were deposited from the column effluent by centrifugation at 200g and suspended, at 37" in minimal pulse label with [3H]uridineindicates that there was less than essential medium (Eagle, 1959) supplemented with 20% au10% nuclear breakage by this technique. tologous plasma, glutamine (0.3 g/l.), penicillin (105 unitsjl.), Rous sarcoma virus 71s RNA labeled with ['*C]uridine, and streptomycin (100 mgil.). In this medium, small lymphowhich was used as a marker, was obtained from transformed cytes were found viable by dye exclusion and capable of chick fibroblast cultures by a previously described method accumulating labeled precursor into RNA in linear fashion (Robinson et al., 1965). for periods of up to 48 hr (Torelli et al., 1968). For [3H]~riAgarose-Acrylamide Gel Electrophoresis of RNA. The dine labeling, 20 pCi/ml of the isotope was added. For 32P method used in this study was adapted from that described by Kapodia et al. (1971). A 1.1 % solution of agarose was prelabeling this isotope, in concentrations of 100 pCi/ml, was added to the cells in phosphate-free media and 2 0 z autolopared by boiling and then cooled to 50" in a temperature gous plasma dialyzed against phosphate-free media. For regulated water bath. For preparation of six gel columns, 5 ml pulse-chase experiments, the cells were deposited from the of acrylamide monomer and bis in twice the final concentramedium containing isotope by centrifugation at 200g for 3 tion required for the gel was mixed with 2.5 ml of cacodylste rnin and resuspended in identical fresh medium containing buffer (0.08 M cacodylic acid-0.04 M NaOH, pH 6.0) and 20 pgiml of cold uridine. Centrifugation in this manner does deoxygenated by stirring for 5 rnin under a vacuum (243 mm). not affect the incorporation of labeled precursors into lymThis solution was heated to 50" in the water bath and 2.25 ml phocyte RNA (Cooper, 1968). The nucleic acid into which of agarose solution was added and mixed. Finally, 0.25 ml isotopic uridine is incorporated by human lymphocytes culof a fresh solution of 0.022 % riboflavin and 0.6 2 ammonium persulfate along with TEMED, 15 ~1 for 2.0% acrylamide tured in the manner described has been clearly identified in previous studies as RNA by sensitivity to alkaline hydrolygels and 30 p1 for 1.76% acrylamide gels, were added. The sis (Torelli era/., 1968), inhibition by actinomycin D (Cooper mixture was pipetted in 1.5-ml aliquots into acid washed, 7 x 10 mm glass columns precoated with 10% photoflow and and Rubin, 1965; Kay, 1967; Neiman and Macdonnell, 1970), insensitivity to DNase (Rubin and Cooper, 1965), and failure fitted with rubber stoppers. In the case of 1.76% acrylamide, 0 , 2 z bis gels the lower half of the columns was also preto bind nitrocellulose filters (Neiman and Henry, 1969). coated with 0.1 agarose to prevent the gels from falling out. Incorporation of [5-3H]uridine into DNA in unstimulated A small layer of hot distilled water was layered above each gel lymphocytes could not be detected (Torelli et al., 1968). column with a long needle and the gels were immersed in the Extraction of R N A . Following incubation, cells were harfilled lower buffer chamber of the electrophoresis apparatus vested by centrifugation at 200g and washed with cold normal saline and the RNA was extracted according to a previously deprecooled to 8"; 1-hr exposure to two opposing fluorescent light sources was allowed for polymerization. Following scribed hot phenol-m-cresol-sodiumdodecyl sulfate procedure gelation, the rubber caps were removed and the upper buffer for sucrose gradient analysis of lymphocyte RNA (Torelli chamber was filled with 0.02 M cacodylate buffer (pH 6.0, identie f ul., 1968). The RNA pellet was washed once with 5 ml of cold 70 ethyl alcohol to remove residual salt and dissolved cal with lower chamber buffer). RNA samples in 10 p1 of water in distilled water (final concentration 5-10 mg of RNA/ml). were mixed with an equal volume of 50% sucrose containing In experiments where very small numbers of cells were exa bromophenol blue dye marker and layered on the upper tracted, unlabeled Escherichia coli RNA was added to the surface of the gel column. The RNA concentrates at the cells as carrier. sucrose gel interface and migrates in the gel toward the cathode under the influence of a current of 3 mA/gel. For experiments involving subcellular fractionation, the procedure was varied as follows: washed lymphocytes were Analysis by Gel Electrophoresis. Bulk species of RNA can suspended in cold hypotonic swelling solution (0.01 M Trisbe localized by staining for 15 min in 0.4% new methylene blue-0.4 M acetic acid-0.4 M sodium acetate followed by 18-hr destaining in cold running water. Examples of stained gels Abbreviations used are: bis, N,N'-methylenebisacrylamide; TEMED, from electrophoresis of E. coli and lymphocyte marker RNAs h',N,.V',.V'-tetramethylefhylenediamine;SSC, standard saline citrate are seen in Figure 1. As has been previously described (Peabuffer, 0.15 ht NaCl-0.015 ht sodium citrate.
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