Isolation and cell-free translation of immunoglobulin messenger RNA

ABCHIVES

OF BIOCHEMISTBY

Isolation

AND

BIOPHYSICS

172, 74-89 (1976)

and Cell-Free Translation

MICHAEL

of lmmunoglobulin

GREEN, TOVA ZEHAVI-WILLNER, JAMES McINNES, AND SIDNEY Roche Institute

of Molecular

Biology,

Nutley,

PETER PESTKA

Messenger

RNA

N. GRAVES,

New Jersey 07110

Received May 27, 1975 The mRNA’s for both the heavy chain (H315) and the light chain (L3i5) of the mineral oil-induced plasmacytoma-315 myeloma protein have been isolated and partially purified from both total cellular RNA and RNA derived from membrane-bound polysomes. The yields of both L315 mRNA and, in particular, of H315 mRNA were increased when total cellular RNA was used as starting material. Total poly(A)-containing mRNA and partially purified mRNA obtained by preparative sucrose gradient sedimentation stimulated protein synthesis in cell-free extracts derived from Ehrlich ascites tumor cells or wheat germ. Cell-free products antigenically and structurally related to both the authentic L315 and H3i5 secreted by intact cells were synthesized in the Ehrlich ascites cell-free system in response to the appropriate mRNA’s. Only the L3i5 mRNA was efficiently translated in the cell-free system derived from wheat germ.

tion theory by direct measurement of the number of immunoglobulin genes present in the cellular DNA in hybridization studies (12-17). The theoretical foundations, predictions, and implications of these theories have been reviewed (18). Additionally, the nucleotide sequence of part of an immunoglobulin light chain mRNA has been reported (19). Such studies may help delineate possible processing or translational control regions in the mRNA sequence. The isolation of additional immunoglobulin mRNA’s from other murine plasmacytoma cell lines is necessary to develop and expand our understanding of immunoglobulin biosynthesis and gene expression. This report describes the partial purification and cell-free translation of the mRNA’s for both the heavy chain (H315)’ and the light chain (L315) of the MOPC315 myeloma protein, an immunoglobulin of the IgA class with antibody-like affinity

The plasmacytoma cell is a neoplastic derivative of the monoclonal, immunoglobulin-producing plasma cell. Murine plasmacytoma cell lines offer a valuable experimental system in which to investigate the origin of antibody diversity, the cellular commitment and specialization of lymphocyte populations, and the intracellular control and organization of immunoglobulin biosynthesis. In recent years, several immunoglobulin mRNA’s from a variety of murine plasmacytoma lines have been isolated and characterized in order to gain an insight into some of these questions (l-9). Standard methods, such as density gradient fractionation and oligo(dT)-cellulose chromatography have been employed to purify these mRNA molecules. In addition, specific techniques, such as the immunoprecipitation of immunoglobulin-synthesizing polysomes (9, 10) or the immunoprecipitation of a complex formed between the immunoglobulin heavy chain mRNA and the complete immunoglobulin molecule have been used (11). These mRNA preparations have been used in initial attempts to distinguish between the major theories pertaining to the generation of antibody diversity, the germ-line theory, the somatic mutation theory, and the somatic recombina-

’ Abbreviations used: MOPC, mineral oil-induced plasmacytoma; IgA315, MOPC-315 protein; H315, heavy chain of MOPC-315 protein; L315, light chain of MOPC-315 protein; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; TPCK, L-l-tosylamido-2-phenylethyl chloromethyl ketone; RPC, adjuvant induced plasmacytoma. 74

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISOLATION

for dinitrophenyl (20).

and trinitrophenyl

EXPERIMENTAL

OF IMMUNOGLOBULIN

groups

PROCEDURES

Chemicals. [SHlleucine (53 Ci/mmol), L3Hlvaline (39 Ci/mmol), t3H]alanine (42 Ci/mmol), YClleucine (348 mCi/mmol), [‘Qvaline (265 mCi/mmol) and YC]alanine (171 mCilmmo1) were obtained from Amersham/Searle. [35S]methionine (200-400 Cii mmol was obtained from New England Nuclear Corp. DNase (electrophoretically purified, RNase free) and trypsin (TPCK-treated) were obtained from Worthington. Oligo(dT)-cellulose was prepared by the method of Gilham (21) and was the generous gift of Dr. S. Kerwar. Solutions. In all the procedures to be described here, all solutions were prepared with autoclaved distilled water and all glassware was autoclaved. Frequently used solutions were designated as follows: Solution I: 10 mM sodium acetate (pH 5.0), 1.2 g/liter of polyvinyl sulfate, 0.3 g/liter of bentonite, 1.0 g/liter of &hydroxyquinoline, 5 g/liter of sodium dodecyl sulfate. SoZutionZZ: 1.0 M Tris-HCl (pH 9.0), 1.0 M NaCl, 0.01 M EDTA. Solution III: 50 mM TrisHCl (pH 7.4), 25 mM KCl, 5 mM MgCl,, 6 mM 2mercaptoethanol. Solution IV: 35 mM HEPES (to pH 7.6 with NH,OH), 146 mM NaCl. Solution V: 10 mM HEPES (to pH 7.6 with NH,OH), 10 mM KCl, 1.5 mM magnesium acetate, 6 mM 2-mercaptoethanol. SoZution VI: 0.2 M HEPES (to pH 7.6 with NH,OH), 1.2 M KCl, 0.05 M magnesium acetate, 0.06 M 2-mercaptoethanol. Solution VII: 20 mM HEPES (to pH 7.6 with NH,OH), 100 mM KCl, 1.0 mM magnesium acetate, 2 mM CaCl%, 6 ml& 2-mercaptoethanol. Tumor lines. MOPC-315 plasmacytoma was obtained from Dr. M. Scharff and Dr. H. Eisen. MOPC-41 plasmacytoma was obtained from Dr. M. Scharff and the RPC-20 and MOPC-300 tumor lines were supplied by Dr. P. Heller. All plasmacytoma lines were maintained as solid tumors in Balb/c mice by serial subcutaneous passage of 0.2-0.3 ml of minced tumor fragments at intervals of lo-15 days. Excised tumors were either used immediately or rapidly frozen in liquid nitrogen and stored in the vapor phase of ,a liquid nitrogen refrigerator. Labeling of intact cells. Preparation and labeling of tumor cell suspensions were performed by minor modifications of previously published procedures (22). [‘4Clleucine, [‘Qvaline, and [14Clalanine were used to label MOPC-315 cell suspensions. The proteins secreted into the medium were used as the source of authentic H3” and L315 radioactive markers. Extraction of total ceZZuZar RNA. Total nucleic acids were extracted from tumor tissue according to minor modifications of previously described methods (23). Tumors were excised and homogenized directly in a mixture of one volume of solution I, one volume

mRNA

75

of redistilled, water-saturated phenol and one volume of chloroform for 2 min at full speed in an Omnimixer (Sorvall). Approximately 15 g of tumor tissue were homogenized in 600 ml of total volume. The homogenate was heated to 56°C and then quickly cooled to 4°C in an ice bath. The mixture was centrifuged for 15 min at 10,000 rpm in a GSA rotor of an RC-2B centrifuge (Sorvall). The upper aqueous layer was removed. The phenol layer was reextracted with 300 ml of solution I. The nucleic acids in the pooled aqueous layers were precipitated by the addition of three volumes of absolute ethanol and stored overnight at -20°C. The precipitate was harvested by centrifugation for 30 minutes at 1500 rpm in a No. 276 rotor of a PR-6 centrifuge (International Equipment Company). The RNA pellet was allowed to drain and then resuspended in water to a concentration of 100-200 A,,, units/ml.2 One-tenth (pH volume of a solution containing 0.1 M Tris-HCl 7.2) and 0.05 M MgCl, was added, followed by the addition of DNase to 10 pg/ml. After incubation at 23°C for 20 min, one-tenth volume of solution II was added and the resulting solution was made 1% in sodium dodecyl sulfate. The RNA was extracted with one volume of a solution of redistilled, watersaturated phenol:chloroform:isoamyl alcohol (50:50: 1, by volume) by vigorous shaking for 10 min at room temperature. The mixture was chilled in an ice bath for 5 min, then centrifuged at 2000 rpm for 30 min in a No. 259 rotor of a PR-6 centrifuge (International Equipment Company). The upper, aqueous layers were pooled and reextracted with another volume of the phenol:chloroform:isoamyl alcohol solution as described above. The RNA in the aqueous fraction was precipitated at -20°C overnight by the addition of one-tenth volume of 20% potassium acetate (pH 5.5) and two volumes of absolute ethanol. The RNA pellet obtained after centrifugation (30 min at 1500 rpm in the No. 276 rotor of the PR-6 centrifuge) was allowed to drain and then the RNA was dissolved in water and stored in the vapor phase of a liquid nitrogen refrigerator until used for oligo(dT)-cellulose chromatography. Preparation of membrane-bound polysomal RNA. T.tembrane-bound polysomes were prepared by minor modifications of described procedures (3, 24). Tumor tissue was disrupted in 0.88 M sucrose in solution III for 30 s at half speed in a Waring blendor. A ratio of 2.4 ml of fluid per g of tumor tissue was used. The tumor fi-agments were then homogenized by eight strokes in a loose-fitting Teflon-glass homogenizer (O.Ol-in. or 0.025cm clearance). The homogenate was centrifuged at 13,000 rpm for 20 min at 4°C in an SS-34 rotor of an RC-2B centrifuge 2 One A,,, unit is the amount of material that in 1.0 ml would yield a value of 1.0 for absorbance at 260 nm in a cuvette with a pathlength of 1.0 cm.

76

GREEN

(Sorvall). The supernatant fluid was removed and diluted to 0.62 M sucrose by the addition of 0.42 volumes of solution III. Each 40-ml portion of the adjusted supernatant fraction was placed onto a twostep sucrose gradient consisting of 17 ml of 1.5 M sucrose in solution III and 13 ml of 2.3 M sucrose in solution III. Centrifugation was performed at 4°C for 16 h at 34,500 rpm in a Type 35 rotor of an L2-65B centrifuge (Spinco). The microsomal band at the interface of the 1.5 and 2.3 M sucrose layers was aspirated and diluted four to five times in water. One-tenth volume of solution II was added, followed by the addition of sodium dodecyl sulfate to a final concentration of 1%. The resulting mixture was extracted with phenol:chloroform:isoamyl alcohol as described above. Oligo(dTkcellulose chromatography. Total cellular RNA (about 3000 AZ80 units) or membrane-bound polysomal RNA (about 1750 A,,, units) at a concentration of 30-50AIlW units/ml in 0.01 M Tris-HCI (pH 7.4) and 0.5 M KC1 was applied to a 1.5 x 6.0~cm column of oligo(dT)-cellulose previously equilibrated with the same buffer. After the sample had entered the column, the column was eluted sequentially at a flow rate of lo-20 ml/h with 50-75 ml of 0.01 M Tris-HCl (pH 7.4) containing 0.5 M KCl, 5075 ml of 0.01 M Tris-HCl (pH 7.4) containing 0.1 M KCl, and 50 ml of 0.01 M Tris-HCl (pH 7.4). Fractions of 6 ml were collected for both the 0.5 and 0.1 M KC1 washes and fractions of 3 ml were collected for the final elution. The absorption at 260 nm of the column fractions was determined and the appropriate fractions were pooled. The RNA was then precipitated by the addition of one-tenth volume of 20% potassium acetate (pH 5.5) and two volumes of absolute ethanol and overnight storage at -20°C. The resulting RNA pellets were dissolved in water and stored in the vapor phase of a liquid nitrogen refrigerator. The oligo(dT)-cellulose column was regenerated by washing with 0.1 M KOH and reequilibrated with 0.01 M Tris-HCl (pH 7.4) containing 0.5 M KCl. Chromatography on oligo(dT)-cellulose was performed at room temperature. Analytical sucrose gradients. RNA (about 0.5AzM) unit) was analyzed on linear 12.5-ml gradients of 520% sucrose (w/v) in 0.02 M sodium acetate (pH 5.0) in an SW 40 rotor of the L2-65B Spinco ultracentrifuge. Centrifugation was performed at 40,000 rpm for 6 h at 4°C and analyzed automatically at 260 nm with a Gilford 2400-S spectrophotometer adapted to an ISCO flow cell (25). Preparative sucrose gradients. Poly(A)-containing mRNA (15-25 A,,, units) derived from either the total cellular RNA or the membrane-bound polysoma1 RNA was fractionated on linear 16.5-ml gradients of 15-30% sucrose (w/v) in 10 mM Tris-HCI (pH 7.4), 100 mr+r NaCl, 1 rnrd EDTA, 0.5% (w/v) sodium dodecyl sulfate in an SW 27.1 rotor. Centrifu-

ET AL. gation was performed at 25,000 rpm for 16 h at 18°C in the L2-65B ultracentrifuge. The gradients were analyzed as described above. The RNA in the appropriate pooled fractions was precipitated by the addition of 2.5 volumes of absolute ethanol and stored overnight at -20°C. The RNA pellet was dissolved in HZ0 and stored in the vapor phase of liquid nitrogen refrigerator. Preparation of Ehrlich ascites S30. An incubated 30,OOOg supernatant fraction (S-30) derived from Ehrlich ascites cells was prepared according to minor modifications of described procedures (26). Ascitic fluid containing minimal amounts of blood was supplied by Miss M. Buck of Roche Laboratories. Cells were washed three times with solution IV by centrifugation at 480 rpm for 10 min at 4°C in a No. 253 rotor of the PR-6 centrifuge (International Equipment Company). After a final centrifugation at 780 rpm for 5 min, cells were resuspended in solution V and allowed to swell for 10 min at 4°C. The swollen cells were disrupted by 25 strokes of the B plunger in a tight-fitting Dounce glass homogenizer (Bellco Glass, Inc.). The final salt concentration of the homogenate was adjusted by the addition of one-tenth volume of solution VI. The homogenate was centrifuged for 10 min at 20,000 rpm in an SS-34 rotor of an RC-2B Sorvall centrifuge. The supematant fluid was carefully aspirated, adjusted to 1 mM ATP, 0.1 mM GTP, 0.6 mM CTP, 10 mM creatine phosphate, 0.2 mg/ml of creatine phosphokinase, and 40 PM in the 20 naturally occurring amino acids, and incubated at 37°C for 1 h. After incubation, low molecular weight components such as amino acids and nucleoside triphosphates were removed by passage through a Sephadex G-25 column or by. dialysis. In addition, the extract was brought to 20 mM HEPES (to pH 7.6 with NHaOH), 120 mM KCI, 5 mM magnesium acetate and 6 mM 2-mercaptoethanol by passage through the column of Sephadex G-25 (coarse) equilibrated with the same solution (or by dialysis against the same solution). The most concentrated column fractions were pooled and the protein concentration determined by the method of Lowry et al. (27). The incubated S-30 was divided into small aliquots and stored in the vapor phase of a liquid nitrogen refrigerator. Ehrlich ascites S-30 preparations of essentially the same activity for cell-free protein synthesis were obtained if Tris buffer was substituted for HEPES buffer or if the incubation step was carried out at 12 mM KC1 instead of 120 mM KCl. Preparation of wheat germ S30. An incubated S30 derived from commercial wheat germ was prepared according to minor modifications of described procedures (28). Raw wheat germ (Niblack) was mixed with an equal weight of ignited sand (Fisher) in a porcelain mortar. The wheat germ was allowed to swell for 1 min in solution VII and then ground

ISOLATION

OF IMMUNOGLOBULIN

gently for 1 min. The homogenate was centrifuged at 4°C for 10 min at 20,000 rpm in the SS-34 rotor of the RC-2B Sorvall centrifuge. The supernatant fluid was carefully aspirated, avoiding the top fatty layer. The supernatant fraction was brought to 3.5 mM magnesium acetate, 1 mM ATP, 0.1 mM GTP, 5 mM creatine phosphate, 40 pg/ml of creatine kinase and 2 mM dithiothreitol and incubated for 15 min at 30°C. The incubated supematant fluid was then passed through a column of Sephadex G-25 (coarse) previously equilibrated with 20 mM HEPES (to pH 7.6 with NH,OH), 5 mM magnesium acetate, 120 mM KC1 and 6 mM 2-mercaptoethanol. The most concentrated column fractions were pooled, divided into small aliquots and stored in the vapor phase of a liquid nitrogen refrigerator. The protein concentration was estimated by the method of Lowry et al. (27). Wheat germ S-30 preparations of similar activity were obtained if Tris buffer was substituted for HEPES buffer or if the incubation was carried out at 23°C instead of 30°C. Preparation o,f rabbit reticulocyte initiation factors. The reticulocyte lysate and the 33-68% (NH&SO, fraction of the 0.5 M KC1 wash of reticulocyte polysomes were prepared by methods similar to those of previously described procedures (29). All procedures were performed at 4°C. Polysomes were removed from the lysate by centrifugation for 120 min at 33,000 rpm in a Type 35 Spinco rotor. The polysome pellet ‘was resuspended in 0.25 M sucrose, 0.1 mM EDTA, and 1 mM dithiothreitol to a final concentration of 125 AzBO units/ml. The suspension was brought to 0.5 M KC1 by the gradual addition of an appropriate volume of 4 M KC1 and stirred for a total of 60 min. Polysomes were then sedimented by centrifugation for 90 min at 50,000 rpm in a Type 65 Spinco rotor. The upper four-fifths of the supematant fluid was removed and brought to 33% saturation by the addit.ion of 0.182 g of solid (NH&SO, per ml. After 1 h of gentle stirring, the precipitate was sedimented by centrifugation at 10,000 rpm for 15 min in the SS-:34 Sorvall rotor. The supernatant fluid was removed and brought to 68% saturation with solid (NH&SO, (an additional 0.220 g/ml) and stirred for an additional hour. The precipitate was sedimented as above and the pellet was washed by resuspension in 10 mM Tris-HCl (pH 7.0), 1.0 mM MgCl,, 100 mM KCl, 0.1 mM EDTA and 1.0 mM 2mercaptoethanol containing (NH&SO4 at 70% saturation. The suspension was centrifuged as above, the supematant fluid was removed, and the pellet was dissolved in a small volume (about 0.01 volume of original lysate) of 10 mM Tris-HCl (pH 7.6), 1.0 mM MgCl,, 100 mM KCl, 0.1 mM EDTA and 1.0 mM dithiothreitol and dialyzed overnight against the same solution. ‘The precipitate that formed during dialysis was rernoved by centrifugation at 5000 rpm for 10 min in the SS-34 Sorvall rotor. The protein

mRNA

77

concentration of the supernatant fraction was determined. The preparation was divided into O.lO-ml portions and stored in the vapor phase of a liquid nitrogen refrigerator. Cell-free protein synthesis with Ehrlich ascites S30. Each 0.050-ml reaction mixture contained the following components: 0.15-0.23 mg of S-30 protein; 70 pg of reticulocyte polysomal salt wash fraction protein; 0.05-0.1 A,,, unit of mRNA; 10 mM TrisHCl (pH 7.5) or 10 mM HEPES (pH 7.5); 120 mM KCl; 3.5 mM MgCl,; 1 mM ATP; 0.1 mM GTP; 5 mM creatine phosphate; 8 pg of creatine kinase; 2 mM 2mercaptoethanol; either 10 /.&i of [Wlmethionine or 10 &i each of [3Hlleucine, L3Hlvaline and [3Hlalanine; and each of the unlabeled amino acids at a concentration of 40 PM. Incubations were performed for 60 min at 30°C. Incorporation of radioactivity into hot trichloroacetic acid-precipitable material was routinely determined on a 5+1 aliquot from the reaction mixture by the method of Mans and Novelli (30). Cell-free protein synthesis with wheat germ S30. Each 0.050-ml reaction mixture contained the following components: 0.40-0.45 mg of S-30 protein; 0.05-0.10 A,, unit of mRNA; 10 mM Tris-HCl (pH 7.5) or 10 mM HEPES (pH 7.6); 120 mM KCl; 1 mM ATP; 0.1 mM GTP; 5 mM creatine phosphate; 8 pg of creatine kinase; 2 mM 2-mercaptoethanol; 70 fig of reticulocyte initiation factors where indicated; and [Y+!]methionine and the complementary unlabeled amino acids as described above for the Ehrlich ascites extract. Incubations were performed for 60 min at 23°C in the absence or at 37°C in the presence of initiation factors. Incorporation of radioactivity was determined as described above for the Ehrlich ascites extract. Gel electrophoresis. Total cell-free products labeled with [35Slmethionine were analyzed on the discontinuous sodium dodecyl sulfate-polyacrylamide-gel system of Laemmli (31) modified for use as a slab gel. Cell-free incubation mixtures were incubated with RNase (10 pg/ml) and EDTA (10 mM) for 15 min at 37°C. Two volumes of gel sample solution containing 62.5 mM Tris-HCl (pH 6.8), 3% (w/v) sodium dodecyl sulfate, 5% 2-mercaptoethanol, and 10% (w/v) glycerol were added and then the mixture was boiled for 2 min. Portions of the boiled samples (30 ~1) were subjected to electrophoresis in a lo-cm x 16-cm x 1.2-mm slab of 12.5% polyacrylamide containing 0.1% sodium dodecyl sulfate at 30 mA (loo200 V during the run) until the bromophenol blue tracker dye reached the bottom of the slab. The gel was removed, fixed, stained, and dried. Autoradiography was performed on RP Royal X-Omat X-ray film (Kodak). Immunoprecipitates of 3H-labeled cell-free products were analyzed on 7.5% continuous, cylindrical sodium dodecyl sulfate gels as described previously

78

GREEN

(22). After electrophoresis, gels were mechanically fractionated (Gel Fractionator, Gilson Medical Electronics) into scintillation vials and counted in Triton X-lOO/toluene scintillation cocktail containing 10% (v/v) water (32). Immunochemical analysis. MOPC-315 myeloma protein was purified from the sera of tumor-bearing mice by minor modifications of described procedures (33, 34). Purified H315 and L315 were subsequently isolated as described (33). Antisera against H315 or L315were raised in rabbits by biweekly injections of 100 pg of either H315 or L315 in Freund’s complete adjuvant. The rabbits developed antibodies to the proteins after 6-8 weeks on this injection schedule. Cell-free reactions with 13H]leucine, 13H]valine and VHlalanine were stopped by the addition of RNase (to 10 cLg/ml) and EDTA (to 10 mM) and incubated at 37°C for 15 min. The samples were brought to 2% sodium dodecyl sulfate, 0.075 M 2-mercaptoethanol and reduced by incubation at 37°C for 60 min. Alkylation was then performed at 37°C for an additional 30 min in the presence of 0.1 M iodoacetamide. The reduced and alkylated samples were dialyzed overnight against 0.1% sodium dodecyl sulfate in 0.01 M sodium phosphate (pH 7.0). The dialyzed samples were used for immunochemical analysis by two procedures. In the first method, portions of the cell-free products containing about 50,000 acid-precipitable cpm were brought to 0.050 ml with 0.15 M NaCl. Nonimmune or immune rabbit serum (2 ~1) was added, followed, after l-5 min, by 10 ~1 of sheep anti-rabbit IgG (Grand Island Biochemical Co.). The samples were then incubated for 45 min at 37°C and allowed to stand overnight at 4°C. One milliliter of 0.01 M sodium phosphate (pH 7.2) and 0.15 M NaCl was added to each tube and the precipitate recovered by centrifugation at 4°C for 30 min at 15,000 rpm in the SS-34 Sorvall rotor. The supematant fluid was carefully removed and discarded. The pellet was dissolved in a small volume (about 0.1 ml) of 0.01 M sodium phosphate (pH 7.0) containing 0.5% (w/v) sodium dodecyl sulfate, 0.14 M 2-mercaptoethanol, and 10% (v!v) glycerol by heating to 90°C for 2 min. The dissolved immunoprecipitates were then subjected to electrophoresis on continuous, cylindrical, 7.5% sodium dodecyl sulfate-polyacrylamide gels as described. Alternatively, the labeled products were first subjected to electrophoresis on the continuous cylindrical gels. The gels were mechanically fractionated and the radioactivity eluted from each gel slice was tested for its ability to be precipitated by either normal, anti-H315, or anti-L315 rabbit serum by indirect immunoprecipitation as described above. Analysis of tryptic peptides. Digestion with trypsin (35) and analysis of the tryptic peptides (36) were performed according to minor modifications of described procedures. H315 and L315 cell-free products labeled with 13H]leucine, 13H]valine and r3H]alanine

ET AL were purified from the total reaction mixture on 7.5% continuous sodium dodecyl sulfate-polyacrylamide gels. Authentic H315 and L315 secreted by intact cells were purified in the same manner. The 3Hlabeled cell-free product and the homologous 14Clabeled cellular product were mixed with 10 mg of IgA315 added as carrier, reduced in 8 M urea and 0.1 M dithiothreitol for 1 h at 37°C and then alkylated with iodoacetamide (0.2 M final concentration) for an additional 45 min at 37°C. Proteins were precipitated by addition of 10 volumes of a solution of cold acet0ne:l.O N HCl (4O:l). After 15 min at 4°C the precipitate was removed by centrifugation at 1500 rpm for 10 min in the PR-6 centrifuge. The pellet obtained was washed twice with 2 ml of ether and dried. Water (300 ~1) was added to the pellet along with enough 2 N NaOH to bring the sample to pH 10. The dissolved pellet was digested in 0.5 ml containing 50 mM Tris-HCl (pH 8.8) and 200 pg of TPCKtreated trypsin for 4.5 h at 37°C with occasional gentle swirling. At the end of the digestion, the sample volume was brought to 1.0 ml with water and the pH adjusted to 2.0 by the addition of glacial acetic acid or concentrated formic acid for L315or H315 digestions, respectively. Insoluble material was removed by centrifugation and the supernatant solution was applied to the column for peptide analysis. Both the H315 and L315 tryptic peptides were anaion-exchange lyzed on a 1.9 x 20-cm Durram-DClA column. For analysis of L315 tryptic peptides, the column was preequilibrated overnight at 55°C with 0.05 N pyridine/acetate (pH 2.4). After application of the sample, the column was eluted at a flow rate of 10 ml/h with a 250-ml gradient of 0.05 N pyridine/acetate (pH 2.4) to 0.5 N pyridine/acetate (pH 3.75). Another 250-ml gradient of 0.5 N pyridine/acetate (pH 3.75) to 2.0 N pyridine/acetate (pH 5.0) followed. Fractions of 2.2 ml were collected, transferred to scintillation vials, dried at 7O”C, and counted in Triton X-lOO/toluene scintillation cocktail containing 10% (v/v) water. The procedure for H3i5 peptide analysis was the same except that formate was substituted for acetate throughout and that about 100 ml of starting buffer was run through the column to elute some peptides before starting the gradient. RESULTS

RNA Extraction and Fractionation The plasmacytoma RNA used in these experiments was obtained by two different procedures (Fig. 1). In the first procedure (Fig. lA), the total cellular RNA was obtained by extracting the entire tumor. This procedure had the advantage of not artifically biasing, qualitatively or quanti-

ISOLATION

OF IMMUNOGLOBULIN

L.

TUMORS I I.

2. 3. 4.

TOTAL

NUCLEIC

I. 2. 3. 4.

TUMOR 2.4ml/R

D.BBM

~

Pam

Suerow

in !GOLUTION

IU

i i%fi?20,w0.;

20 Min.

SUPERNAlANY

(no)

(520)

I

DILUTE TO 0.62M Sucrora in SOLUTION III

ACIDS

SYBF GRADIENT

DISSOLVE IN H20 ADD 0.1 VOLUME O.IM TRIS-HCI, pH 7.2, 0.05M yIcI2 ADD DNArq TO 10 &ml INCUBATE 15.20 MINUTES AT 23-C

I.51

Sucmr

in SOLUNON lB+

2.3M Sucroa~ in SCUmON IN+

w u CENTRImE 94.000~9, 16 HR

I DNASE

TREATED NUCLEIC I. 2. 3.

I TOTAL

6.

ADD I VOLUME SOLUTION I (pli 5.0) I VOLUME PHENOL, I VOLUME CHLOROFORM HOMOGENIZE HEAT TO 56-C. THEN COOL TO 4-Z CENTRIFUGE FOR AWEOUS LAYER

79

mRNA

ACIDS

I

ADD 0.1 YOLUME SOLUTION II (pH 9.0) ADD 0.1 VOUlME 10% SDS EXTRACT 2X WITH I VOLUME PHENOL CHLOROFORMz im-AMY1 ALCO4iOL (50:5&l by rolumd CELLULAR

RNA

FIG. 1. Flow diagrams bound polysomal RNA.

depicting

RNA

EXTRACTION

I. DILUTE MR POlY5 4X WlTN H20 2. ADD 0.1 WlJMZ 5OlUTION II (rn 9.01 3. ADD 0.1 VWME 10% SDS 4. EXTRACl 2X WlTH I %lUJME PHENOL: CHLOROFORM: iso.AMYl ALCOHOL (50:5&l by roluma) s MEMBRANE-BOUND POLYSOMAL RNA

preparation

tatively, the composition of the mRNA fraction that was subsequently obtained. In the second procedure (Fig. lB1, polyribosomal RNA was extracted from the membrane-bound polysomes recovered as a distinct band in the sucrose step-gradient employed for polysome fractionation. This method attempted to take advantage of the observation made by several workers that immunoglobulin biosynthesis takes place predominantly on ribosomes bound to the endoplasmic reticulum (24, 37, 38). In initial studies, RNA from the plasmacytoma lines MOPC-41, RPC-20, MOPC-300, and MOPC-315 was obtained by the procedure shown in Fig. 1B. Subsequent work, however, on the mRNA’s isolated from MOPC-315 employed both extraction procedures. The poly(A)-containing mRNA fractions were obtained by chromatography of the membrane-bound polysomal RNA or the total cellular RNA on oligo(dT)-cellulose (Fig. 2A). Immunoglobulin mRNA’s, like

of (A) total cellular

Jt

MEMBRANEBOUND POLYSOMES FREE FOLYSOMES

RNA and (B) membrane-

most eucaryotic mRNA’s contain a poly(A) segment at their 3’-hydroxyl end and thus bind to oligo(dT)-cellulose at the appropriate ionic strength (3, 5). The poly(A)-conmining fraction that eluted with the lowest ionic strength buffer usually comprised l-2% of the total material applied to the column. This was the case whether membrane-bound polysomal RNA or total cellular RNA was used. The column fractions were pooled as indicated in Fig. 2A and then concentrated by ethanol precipitation. Analysis of the RNA fractions in sucrose gradients (Fig. 2B) demonstrated that the fraction composed of the sample front and the 0.5 M KC1 eluate contained tRNA and the 5, 18 and 28s rRNA, whereas the 0.1 M KC1 wash fraction contained primarily 18 and 28s rRNA. In initial experiments with oligo(dT)-cellulose chromatography, significant contamination of the poly(A)-containing fractions by rRNA was observed despite lengthy washing of the column with 0.5 M KCl. It was

GREEN

0

ET AL.

~,(J 1:;w-;-^.: I II 20 30 IO FRACTION NUMBER

40

ml

FROM

TOP

OF GRADIENT

Fkc. 2. Oligo(dT)-cellulose chromatography of membrane-bound polysomal RNA from MOPC-315 tumors. (A), Column profile obtained with 1750Azw units of RNA. Elution with 0.5 M KC1 in 0.01 M Tris-HCl (pH 7.4), 0.1 M KC1 in 0.01 M Tris-HCl (pH 7.41, and 0.01 M Tris-HCl (pH 7.4) is indicated by the arrows. Column fractions were pooled for further analysis as indicated by the horizontal bars. (B), Analytical sucrose gradient sedimentation of RNA in pooled column fractions. RNA was analyzed on linear 5-20% sucrose (w/v) gradients in 0.02 M sodium acetate (pH 5.0). Centrifugation was performed at 40,000 rpm for 6 h at 4°C in an SW 40 rotor of an L2-65B Spinco ultracentrifuge.

discovered, however, that introduction of the 0.1 M KC1 wash step prior to the final elution removed most of the residual rRNA bound to the column. The major component of this residual RNA is 28s rRNA (Fig. 2B). The poly(A)-containing mRNA fraction obtained from the oligo(dT)-cellulose column in the final elution step is a mixture of several RNA species (Fig. 2B). A convenient further fractionation of both the poly(A)-containing mRNA derived from the total cellular RNA and that derived from membrane-bound polysomal RNA was performed on preparative sucrose gradients containing sodium dodecyl sulfate (Fig. 3). The sedimentation profiles of the two types of mRNA preparations were quite different. It is evident that the total cellular poly(A)-containing mRNA fraction (Fig. 3A) contains more of the RNA species sedimenting at 16-19s as well as a greater fraction of heavier RNA species sedimenting at 22-288. The gradient fractions were pooled as indicated in Fig. 3, and the RNA was recovered by ethanol precipitation. The precipitated RNA was dissolved in water and stored in the vapor phase of a liquid nitrogen refrigerator until used for cell-free translation studies.

Cell-free Translation

of mRNA.

The poly(A)-containing mRNA fractions obtained from oligo(dT)-cellulose chromatography (Fig. 2) and sucrose gradient sedimentation (Fig. 3) were tested for their ability to stimulate the synthesis of protein in cell-free extracts derived from either Ehrlich ascites tumor cells (Table I) or wheat germ (Table II). The stimulation of protein synthesis in the Ehrlich ascites S-30 extract by immunoglobulin mRNA was enhanced in the presence of the 3368% (NH&SO4 fraction of the 0.5 M KC1 reticulocyte polysomal wash. Previous work from other laboratories with an S-30 extract prepared from Krebs II ascites cells did not demonstrate a necessity for supplementation of the cell-free system with this initiation factor preparation (3, 39). The reason for this difference is at present unknown. The supplemented ascites system is quite sensitive; as little as 0.02-0.05 AzsO unit of mRNA per 0.050-ml reaction mixture yielded detectable incorporation of labeled amino acids into specific polypeptides. Although the ascites system responded to small amounts of exogenous mRNA, it had the disadvantage of possessing a relatively high level of endogenous activity

ISOLATION

OF IMMUNOGLOBULIN

81

mRNA

FIG. 3. Preparative sodium dodecyl sulfate-sucrose gradient fractionation of poly(A)-containing mRNA extracted from MOPC-315 tumors. (A), Poly(A)-containing mRNA from total cellular RNA; (B), poly(A)-containing mRNA from membrane-bound polysomal RNA. The gradients were 15-308 sucrose (w/v) in 0.01 M Tris-HCl (pH 7.4), 0.1 M NaCl, 0.01 M EDTA, and 0.5% sodium dodecyl sulfate. Centrifugation was performed at 18°C for 16 h at 26,000 rpm in a Spinco SW 27.1 rotor. Fractions were pooled as indicated. The positions of 5, 18, and 28s rRNA obtained on a parallel gradient are indicated. TABLE

I

CELL-FREE PROTEIN SYNTHESIS IN EHRLICH ASCITES S-30 WITH IMMUNOGLOBULIN RNA [3H]leucine

RNA added (AA,, units/O.05 ml)

incorporated

Minus reticulocyte ribosomal wash factors None MOPC-315 membrane-bound poly(A) mRNA (0.05) MOPC-315 membrane-bound 0.5 M KC1 wash RNA (0.1) MOPC-41 membrane-bound poly(A) mRNA (0.05) MOPC-41 membrane-bound 0.5 M KC1 wash RNA (0.2) RPC-20 membrane-bound poly(A) mRNA (0.05) RPC-20 membrane-bound 0.5 M KC1 wash RNA (0.1) LIIncubation ribosomal wash mRNA fraction first panel, Fig. mRNA fraction

0.30 0.64 0.47 0.75 -

FRACTIONS” (pmol/0.05 ml)

Plus reticulocyte ribosomal wash factors 1.90 6.64 4.33 10.4 7.57 4.76 2.64

conditions are described in Materials and Methods. Where indicated, 70 pg of reticulocyte factors were added. Membrane-bound poly(A) mRNA refers to the poly(A)-containing obtained after oligo(dT)-cellulose chromatography of membrane-bound polysomal RNA (see 2B). The value in parentheses after each RNA fraction refers to the number ofAzeo units of used in each 0.05-ml reaction mixture.

that could possibly complicate experimental findings. For examnle, when the oligo(dT)&llulose 0.5 M -KC1 wash was tested in this system, stimulation of incorporation of radioactive label into hot trichloroacetic acid-precipitable material was observed (Table I). This was somewhat puzzling, since this RNA fraction should contain relatively little, if any, mRNA. However, upon electrophoretic analysis of the cell-free product it was observed that only the addition of the poly(A)-containing mRNA fractions yielded

new protein products. The addition of the oligo(dT)-cellulose 0.5 M KC1 wash fraction seemed to stimulate the synthesis of proteins which corresponded to endogenous activity of the S-30 extract. The wheat germ S-30 extract had a low endogenous background activity; addition of exogenous immunoglobulin mRNA fractions stimulated protein synthesis (Table II). Additionally, the activity of this system could be enhanced in the presence of the 33-68% (NH&SO, fraction of the 0.5 M KC1 reticulocyte polysomal wash and sub-

82

GREEN TABLE

ET AL. II

CELL-FREE PROTEIN SYNTHESIS IN WHEAT GERM S-30 WITH IMMUNOGMBULIN RNA added (Az6,, units/O.05 ml)

13YSlmethionine Minus reticulocyte ribosomal wash factors

None MOPC-315 membrane-bound MOPC-41 membrane-bound

poly(A1 mRNA (0.05) poly(A) mRNA (0.05)

0.03 0.09 0.08

RNA FRACTIONS~

incsrtion

(pmoU0.05 Plus reticulocyte ribosomal wash factors 0.03 0.12 0.15

a Incubation conditions are described in Materials and Methods. Where indicated, 70 fig of reticulocyte ribosomal wash fraction was added. In the absence of factors, MgCl, was present at 2.5 rnru and incubation was performed at 23°C for 60 min. In the presence of factors, MgCl, concentration was 3.5 mM and incubations were performed at 37°C for 60 min. Membrane-bound poly(A) mRNA refers to the poly(A)containing mRNA fraction obtained after oligo(dT)-cellulose chromatography of membrane-bound polysoma1 RNA (see third panel, Fig. 2B). The value in parentheses after each RNA fraction refers to the number of A,, units of mRNA fraction used in each 0.05-ml reaction mixture.

derived from the MOPC-315 membranebound polysomal RNA. Although the pL315 band was evident, the total poly(A)-containing mRNA fraction did not direct the synthesis of detectable amounts of the H315 protein species. This species can be observed in the cell-free products, however, after further fractionation of the total Analysis of Cell-Free Products mRNA (Fig. 4). Preliminary analyses of the protein prodWhen the RNA fraction sedimenting ucts synthesized in response to both the faster than the 16-19s species was tested MOPC-315 poly(A)-containing mRNA frac- in the Ehrlich ascites cell-free system, tions obtained from oligo(dT)-cellulose and both the pL315 and H315 bands were obthe more purified MOPC-315 mRNA frac- served in the products (Fig. 4). This tindtions subsequently obtained from sucrose ing could be attributed to trailing of the gradient sedimentation in the presence of RNA species in the gradient. It is also sodium dodecyl sulfate were performed by possible, however, that the cosedimentaelectrophoresis in polyacrylamide slab- tion of the two activities was due to aggregels. In the Ehrlich ascites system (Fig. 41, gation of the RNA molecules. Aggregation the total poly(A)-containing mRNA de- has been reported for immunoglobulin rived from total cellular RNA directs the mRNA’s extracted by procedures similar synthesis of several major protein species, to those employed here (9). Indeed, it has including one migrating slightly more been observed that incubation of the heavrapidly than authentic H315 and one mi- ier RNA species (Fraction D, Fig. 2A) at grating slightly more slowly than authen80°C for 30 s followed by quick cooling tic L315(pLBIJ in Fig. 4). The 12-14s mRNA resulted in the appearance of both the 12fraction (Fraction B, Fig. 3A) directed the 14 and the 16-19s RNA species in the sedisynthesis of the pL315polypeptide while the mentation profile. 16-19s mRNA fraction (Fraction C, Fig. The cell-free system derived from wheat 3A) directed the synthesis of both the H315 germ differed from the ascites system in its and pL315protein species. The results were response to the immunoglobulin mRNA different in the case of the mRNA fractions fractions. The pL315 protein species was synthesized in the wheat germ S-30 ex3 Zehavi-Willner, T. and Pestka, S., manuscript in preparation. tract in the presence of the total poly(A)sequent incubation of the reaction at 37°C instead of the usual 23°C incubation temperature. This hybrid cell-free system has been employed in some of the experiments presented in this report. A more detailed description of this observation is in preparation.3

ISOLATION

OF IMMUNOGLOBULIN

83

mRNA

rived from wheat germ from different sources (40). However, it is also possible that the H315 protein product was synthesized in amounts too low to be detected by present procedures. The cell-free products synthesized in

FIG. 4. Autoradiogram of [YS]methionine-labeled cell-free products synthesized in the ascites cell-free system and analyzed on sodium dodecyl sulfate-polyacrylamide gel slabs. Products were synthesized in the presence of the following MOPC315 RNA fractions: (1) Total poly(A)-containing mRNA from total cellular RNA (see Fig. 3A); (2) Fraction B, Fig. 3A; (3) Fraction C, Fig. 3A; (4) Fraction D, Fig. 3A; (5) Fraction B plus Fraction C, Fig. 3A; (6) W-labeled H315 and L315 marker proteins; (7) no mRNA; (8) Fraction B’, Fig. 3B; (9) Fraction C’, Fig. 3B; (10) total poly(A)-containing mRNA from membrane-bound polysomal RNA; (11) ‘%-labeled H315 and L315 marker proteins. The positions of authentic H315 and L315 as well as the position of the L315 precursor (pL315) are indicated.

containing mRNA as well as in the presence of the 12-14s (Fraction B, Fig. 3A) and 16-19s (Fraction C, Fig. 2A) sodium dodecyl sulfate-sucrose gradient fractions (Fig. 5). It appeared, however, that the H315polypeptide was not efficiently synthesized even in response to the 16-19s sodium dodecyl sulfate-sucrose gradient fraction. This observation contrasts with the results of others who observed possible immunoglobulin heavy chain synthesis in an S-30 extract derived from wheat germ (2). This difference may be attributed to the reported variation in the activities of cell-free protein-synthesizing systems de-

12

3

456

FIG. 5. Autoradiogram of [Wlmethionine-labeled cell-free products synthesized in the wheat germ cell-free system in the presence of reticulocyte initiation factors and analyzed on sodium dodecyl sulfate-polyacrylamide gel slabs. Products were synthesized in the presence of the following MOPC315 RNA fractions: (1) W-labeled H315 and L315 marker proteins; (2) no mRNA; (3) total poly(A)containing mRNA from total cellular RNA (see Fig. 3A); (4) Fraction B, Fig. 3A; (5) Fraction C, Fig. 3A; (6) Fraction D, Fig. 3A. The positions of authentic H315 and L315 as well as the position of the L315 precursor (PLY are indicated.

a4

GREEN

both the ascites and wheat germ systems contained polypeptides smaller than L315 as well as the putative L315or H315protein chains (Figs. 4 and 5). The possibility was considered that some of these polypeptides might represent fragments of either H315 or L315. The synthesis of such fragments had been described in studies examining the cell-free translation of immunoglobulin light chain mRNA’s (3, 9). It was hypothesized that these fragments resulted from either premature termination of translation or enzymatic degradation in the cell-free system. Experiments designed to examine the synthesis of the initial peptides of the H315or L315cell-free product should help to elucidate this question. Alternatively, these products could represent proteins coded for by mRNA’s other than H315 or L315 mRNA. Specifically, recent findings indicate that the major band migrating approximately halfway between L315 and the dye front on sodium dodecyl sulfate-polyacrylamide gels represents the product coded for by an mRNA or class of mRNA’s smaller than L315 mRNA as measured by sodium dodecyl sulfate-sucrose sedimentation. A rate of migration similar to an authentic marker protein is suggestive but not proof of identity of protein chains. More stringent tests of identity would include the demonstration that the cell-free products possess the antigenic determinants of the cellular immunoglobulin chains and contain the same primary sequence of the authentic proteins. Figure 6 shows the results of one type of immunochemical analysis of the protein products synthesized in response to the 16-19s gradient fraction (Fraction C, Fig. 3A) of the poly(A)-containing mRNA derived from MOPC-315 total cellular RNA. Normal rabbit serum precipitated little of the radioactivity in the gel slices. The anti-H315 serum precipitated the H315component and some radioactivity present in smaller polypeptide chains. The anti-L315 serum precipitated the pL315 component and a minute amount of the H315 component. The anti-H315 serum used in this experiment was completely specific as measured by double diffusion in Ouchterlony plates and by precip-

ET AL.

6-

0

1 IO

I

I I I , I 20 30 40 FRACTION NUMBER

I

I 50

FIG. 6. Immunochemical analysis of cell-free products of MOPC-315 16-19s mRNA obtained from total cellular RNA (Fraction C, Fig. 2A). Cell-free products synthesized in the ascites cell-free system were fractionated on continuous sodium dodecyl sulfate-polyacrylamide gels. The gels were mechanically fractionated, and the radioactivity eluted from each gel slice was tested for its ability to be precipitated by either normal, anti-H315 or anti-L315 rabbit serum. (A), Immunoprecipitation of gel-slice eluates by normal serum (O), anti-Hal5 serum (0) or antiL315serum (A). (B), Total radioactivity in gel slices.

itation of authentic radioactive L315 and H315under conditions comparable to those of Fig. 6. Thus, the smaller polypeptides that were precipitated are probably fragments of H315 produced during cell-free translation. The anti-L315 serum has been shown to contain a small amount (~5%) of anti-H315 activity thus accounting for the small fraction of heavier component precipitated. The results presented in Fig. 6 are consistent with the identity of the heavier and lighter products with authentic H315 and L315, respectively. Further demonstration of the chemical identity of the cell-free products was given by analysis of the peptides produced by tryptic digestion. The H315 and pL315 polypeptides present in the cell-free products synthesized in the Ehrlich ascites extract in response to MOPC-315 mRNA fractions were purified from the total reaction mix-

ISOLATION

ture,

OF IMMUNOGLOBULIN

with authentic radioactive respectively, and then diThe chromatographic gested with &psin. profiles of both the L315 (Fig. 7) and H315 (Fig. 8) tryptic peptides showed correspondence between the peaks derived from the cell-free product and those derived from the cellular protein. These data and the results obtained from both immunochemical and electrophoretic analysis are consistent with the ,identity of the major cell-free products as the primary products directed by the L315 mRNA and the H315 mRNA of the MOPC-31.5 plasmacytoma. The observation that the primary product of the L3*” mRNA is a longer precursor form of L315 (pL315) is consistent with the data of others pertaining to other immunoglobulin light chains (l-6). Additionally, the fact that the H315mRNA cell-free product migrates slightly faster than authentic H315 is in agreement with the findings of other laboratories (2, 7). It has been postulated that the cell-free products of immunoglobulin heavy chain mRNA’s also contain a precursor segment not found in mature heavy chains but are devoid of the carbohydrate residues contained in authentic secreted heavy chain (7, 41). These two factors would balance each other and result in similar rates of migration for the cellular and cell-free product. Finally, the preliminary estimates of the size of the putative L315 mRNA (12-14s) and H315 mRNA (16-19s) obtained in sodium dodecyl sulfate-sucrose gradients are in agreement with measurements made on other immunoglobulin heavy and light chain mRNA’s U-11). H315

mixed

or

L315

DISCUSSION

The experiments described in this report present studies of the isolation and cellfree translation of MOPC-315 immunoglobulin light chain and heavy chain mRNA’s. These studies have the potential for increasing the scope of cell-free translational analysis of immunoglobulin mRNA’s. The MOPC-315 mRNA’s represent the mRNA’s for a light chain of the hz subgroup and a heavy chain of the (Yclass. These species of immunoglobulin chains have not been as extensively studied as others in cell-free

mRNA

85

protein-synthesizing systems. In addition, cell-free translation of MOPC-315 mRNA’s offers the possibility of investigating the relationship and coordination, if any, between the synthesis of homologous light and heavy chains of a complete immunoglobulin molecule. Furthermore, the fact that the MOPC-315 myeloma protein secreted by intact cells possesses an antibody-like affinity for dinitrophenyl and trinitrophenyl groups provides the potential for the biological assay of cell-free products directed by these mRNA’s. The isolation and characterization of these mRNA’s has also produced several simple, but significant, observations generally applicable to future attempts to isolate other eucaryotic mRNA’s. The general strategy of many workers attempting to isolate the mRNA for a particular immunoglobulin chain has been to derive the mRNA fraction from membrane-bound polysomal RNA, thus obtaining an RNA preparation enriched for these mRNA’s. Although the experimental basis for this is sound, we have shown that such manipulation resulted in a lower yield of immunoglobulin mRNA’s. A comparison of the mRNA fractions derived from MOPC-315 total cellular RNA and membrane-bound polysomal RNA showed that the latter is particularly deficient in the H315 mRNA and heavier RNA species. Such a situation could, of course, result from selective degradation of the RNA’s in the polysome fractionation step. It could also be explained, however, by a selective loss of these RNA’s into cellular fractions other than the microsomal fraction. Some of the RNA that was contained in the total cellular RNA was no doubt derived from the nucleus and thus would be absent from the microsomal fraction. Alternatively, a portion of the additional RNA obtained in the total cellular RNA fraction may be derived from large fragments of rough endoplasmic reticulum that may sediment with the nuclei during the preparation of the postmitochondrial supernatant fraction. Such a situation was observed during the isolation of polysomes synthesizing thyroglobulin (42). If the H315mRNA was contained in such large membrane fragments

86

GREEN

FRACTION

ET AL.

NUMBER

FIG. 7. Chromatography of the tryptic peptides obtained by digestion of a mixture of “Clabeled L315 secreted by intact cells (0) and 3H-labeled L315 precursor (pL39 from the ascites system cell-free products (A) synthesized in the presence of MOPC-315 total poly(A)-containing mRNA extracted from membrane-bound polysomes. The pL315 product was purified from the total cell-free products by fractionation on continuous sodium dodecyl sulfate polyacrylamide gels prior to digestion with trypsin.

after disruption of the tumor tissue, it would sediment with the nuclear pellet, resulting in a selective loss of heavy chain mRNA. Experiments analyzing the distribution of immunoglobulin mRNA’s in the various cellular fractions should provide further insight into this question. The loss of H315mRNA seems to affect the relative efficiency with which the H315 mRNA is translated in a mixture of mRNA species. It appears that when the ratio of H31s mRNA to L315 mRNA is increased, as the case with the 16-19s mRNA fraction obtained from microsomal RNA, H315mRNA is translated in the ascites S-30 cell-free system. One explanation of this phenomenon may be that H315 mRNA is intrinsically a poorer message than L315 mRNA, and may have, for example, a lower absolute rate of initiation. Consistent with this is the observation that the deficiency in H315 mRNA activity in the membranebound mRNA fraction may be overcome by partial purification of the H315 mRNA.

This concept, if supported by the appropriate experiments, could have relevance for the control of immunoglobulin synthesis in intact cells. Alternatively, other factors not delineated here may be necessary for efficient H315 mRNA translation in the presence of competing mRNA’s. The fact that H315 mRNA competed poorly with L315 mRNA for translation in two cell-free systems suggests the need for partial purification of this mRNA for efficient translation. The observation that H315mRNA is not efficiently translated by wheat germ S-30 emphasizes the necessity of testing mRNA preparations in at least two, and perhaps more, cell-free translation systems. The apparent variation in the activity and efficiency of the cell-free translation systems among different laboratories suggests caution in the use of any one of them to analyze the ability of a particular mRNA to direct the synthesis of a specific product. The mRNA fractions described in this

IO

20

30

40

50

60

70

SO

90

100

110

120

FRACTION

130

150

160

NUMBER

140

170

I60

190

XK,

210

220

230

240

250

260

’

-3 - 2.5

I5

FIG. 8. Chromatography of the tryptic peptides obtained by digestion of a mixture of ‘Glabeled H315 secreted by intact cells (0) and putative 3H-labeled H315 from the ascites system cell-free products (LI) synthesized in the presence of MOPC-315 16-19s mRNA derived from total cellular RNA (Fraction C, Fig. 3A). The H315 product was purified from the total cell-free products by fractionation on continuous sodium dodecyl sulfatepolyacrylamide gels prior to digestion with trypsin.

0

6

~,~!!!‘!!!!!!IITIIfI_I

88

GREEN

report are certainly not yet composed of a single species. It should be possible, however, with presently available methods both to estimate the purity of these RNA fractions and to obtain more highly purified L315 and H315 mRNA preparations. When further purified, the mRNA species described in this report can be used in hybridization studies to evaluate and perhaps expand the work of others describing the measurement of immunoglobulin gene frequency. Such studies employing the H315and L315mRNA’s will have the added dimension of allowing the quantitation of both the heavy and light chain genes for a complete immunoglobulin molecule produced by a specific cell line. The purification of these mRNA’s is also the first step in an attempt to examine several questions pertaining to the control and intracellular organization of immunoglobulin biosynthesis as well as that of other secreted proteins. The availability of purified immunoglobulin mRNA preparations will allow a detailed study into the molecular mechanisms underlying the well-documented functional distinction between membranebound and free polysomes as the sites of synthesis of secreted and intracellular proteins, respectively. It should, for example, be possible to examine what functions, if any, the precursor segment of the nascent immunoglobulin chain fulfills in the attachment of heavy and light chain-synthesizing polysomes to the endoplasmic reticulum. It has been suggested that such precursor segments are signals for ribosome membrane interaction common to all proteins whose synthesis is localized on the endoplasmic reticulum (4,43). In addition, in intact cells, the MOPC-315 IgA protein is secreted in the form of dimers joined by a third protein species, the J chain (44). The cell-free synthesis and genetic representation of the J chain should also be of interest. Finally, purified immunoglobulin mRNA’s such as the H315 or L315 mRNA’s for the MOPC-315 IgA molecule can provide the basis for the development of a cellfree system capable of the complete synthesis of an active antibody molecule. Such a

ET AL.

system could provide insights into the detailed mechanisms of ribosome-membrane interaction, carbohydrate addition, and other posttranslational modifications and the role of each of these processes in the synthesis, assembly and secretion of immunoglobulin molecules. REFERENCES 1. STAVNEZER, J., AND HUANG, R. C. C. (1971) Nature New Biol. 230, 172-176. 2. SCHMECKPEPER, B. J., CORY, S., AND ADAMS, J. M. (1974) Mol. Biol. Rep. 1, 355-363. 3. SWAN, D., AVIV, H., AND LEDER, P. (1972)Proc. Nat. Acad. Sci. USA 69, 1967-1971. 4. MILSTEIN, C., BROWNLEE, G. G., HARRISON, T. M., AND MATHEWS, M. B. (1972) Nature New Biol. 239, 117-120. 5. MACH, B., FAUST, C., AND VASSALLI, P. (1973) Proc. Nat. Acad. Sci. USA 70,451-455. 6. TONEGAWA, S., AND BALDI, I. (1973) Biochem. Biophys. Res. Commun. 51, 81-87. 7. COWAN, N. J., AND MILSTEIN, C. (1973) Eur. J. B&hem. 36, 1-7. 8. HARRISON, T. M., BROWNLEE, G. G., AND Mm STEIN, C. (1974) Eur. J. B&hem. 47,621-627. 9. SCHECHTER, I. (1973) Proc. Nat. Acad. Sci. USA 70, 2256-2260. 10. DELOVITCH, T. L., BOYD, S. L., TSAY, H. M., HOLME, G., AND SEHON, A. H. (1973) B&him. Biophys. Acta 299, 621-633. 11. STEVENS, R. H., AND WILLIAMSON, A. R. (1973) Proc. Nat. Acad. Sci. USA 70, 1127-1131. 12. TONEGAWA, S., STEINBERG, C., DUBE, S., AND BERNARDINI, A. (1974) Proc. Nat. Acad. Sci. USA 71, 4027-4031. 13. PREMKUMAR, E., SHOYAB, M., AND WILLIAMSON, A. R. (1974) Proc. Nat. Acad. Sci. USA. 71, 99-103. 14. DELOVITCH, T. L., AND BAGLIONI, C. (1973)Proc. Nat. Acad. Sci. USA 70.173-178. 15. HONJO, T., PACKMAN, S., SWAN, D., NAU, M., AND LEDER, P. (1974) Proc. Nat. Acad. Sci. USA 71, 3659-3663. 16. STAVNEZER, J., HUANG, R. C. C., STAVNEZER, E., AND BISHOP, J. M. (1974) J. Mol. Biol. 88, 43-63. 17. BABBITTS, T. H., BISHOP, J. P., MILSTEIN, C., AND BROWNLEE, G. G. (1974) FEBS Lett. 40, 157-160. 18. SMITH, G. P. HOOD,L., AND FITCH, W. M. (1971) Annu. Rev. B&hem. 40, W-1012. 19. BROWNLEE, G. G., CARTWRIGHT, E. M., COWAN, N. J., JARVIS, J. M., AND MILSTEIN, C. (1973) Nature New Biol. 244, 236-240. 20. EISEN, H. N., SIMMS, E. S., AND POTTER, M. (1968) Biochemistry 7,4126-4134.

ISOLATION 21. GILHAM,

OF IMMUNOGLOBULIN

P. (1964) J. Amer. Chem. Sot. 86,

4982-4985. 22. LASKOV, R., AND SCHARFF, M. D. (1970) J. Exp. Med. 131, 515-542. 23. BHOOPALAM, N., YAKULIS, V., AND HELLER, P. (1973) Clin. Exp. Immunal. 16, 243-258. 24. CIOLI, D., AND LENNOX, E. S. (1973) Biachemistry 12, 3211-3217. 25. PF.STKA, S., AND HINTIKKA, H. (1971) J. Biol.

Chem. 246, 7723-7730. 26. MATHEWS, M. B., AND KORNER, A. (1970)Eur. J. B&hem. 17, 328-338. 27. LOWRY, 0. II., R~SEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 28. ROBERTS, B. E., AND PATERSON, B. M. (1973)

Proc. Nat. Acad. Sci. USA 70, 2330-2334. 29. SHAFRITZ, D. A., DRYSDALE, J. W., AND ISSE~ BACHER, K. J. (1973) J. Biol. Chem. 248,32203227. 30. MANS, R. J., AND NOVELLI, G. D. (1961) Arch.

Biochem. Biophys. 94,48-53. 31. LAEMMLI, IJ. K. (1970) Nature (London) 227, 680-685. 32. PATPERSON, M. S., AND GREENE, R. C. (1965)

Anal. Chem. 37, 854-857. 33. UNDERW~N, B. J., SIMMS, E. S., AND EISEN, H. N. (1971) Biochemistry 10,4359-4368.

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34. G~ETZL, E. G., AND METZGER, H. (1970) Biochemistry 9, 1267-1278. 35. DUGAN, E. S., BRADSHAW, R. A., SIMMS, E. S., AND EISEN, H. N. (1973) Biochemistry 12, 5400-5616. 36. BRADSHAW, R. A., BABIN, D. R., NOMATO, M., SRINIVASIN, N. G., ERICSSON, L. H., WALSH, K. A., AND NEURATH, H. (1969) Biochemistry 8, 3859-3871. 37. LISOWSKA-BERNSTEIN, B., LAMM, M. E., AND VASSALLI, P. (1970) Proc. Nat. Acad. Sci. USA 66, 425-432. 38. MACH, B., KOBLET, H., AND GROS, D. (1968)

Proc. Nat. Acad. Sci. USA 59,445-457. 39. JACOBS-IARENA, M., AND BAGLIONI, C. (1972) Biochemistry 11.4970-4974. 40. ROBERTS, B. E., MATHEWS, M. B., AND BRUTON, C. J. (1973) J. Mol. Biol. 80, 733-742. 41. COWAN, N. J., HARRISON, T. M., BROWNLEE, G. G., AND MILSTEIN, C. (1973) Biochem. Sot. Trans. 1, 1247-1250. 42. VASSART, G., AND DUMONT, J. E. (1973) Eur. J.

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32, 322-330.

43. BLOBEL, G., AND SABATINI, D. D. (1972) in Biomembranes (Manson, L. A., ed.), Vol. 2, pp. 193-195, Plenum, New York. 44. PARKHOUSE, R. M. E. (1972) Nature New Biol. 236,9-11.