Initiation and translation in vitro of mRNA for MOPC 315 immunoglobulin heavy chain and characterization of translation product.

An initiation study of mineral oil-induced plasmacytoma (MOPC) 315 heavy chain immunoglobulin (H315) in vitro has been conducted using formyl-[35S]methionyl-tRNAfMet and a highly purified 18 S message from MOPC 315 solid tumor in a crude rabbit reticulocyte lysate system. The product was specifically precipitated by antibodies directed against MOPC 315 immunoglobulin and H315. The in vitro H315 products terminally labeled with formyl-[35S]methionine or internally labeled with [3H]leucine were electrophoretically identical with in vivo H315 on sodium dodecyl sulfate-polyacrylamide gels. All of the [35S]-methionine was incorporated at the NH2 terminus, not internally, since there is a near complete recovery of [35S]methionine following one cycle of Edman degradation. The NH2-terminal cyanogen bromide peptide, CN2, of in vivo and in vitro H315 co-migrated exactly on gel electrophoresis under conditions which completely resolved two proteins differing in size by only 14 amino acids. These data strongly suggest that there is no NH2-terminal precursor of H315 in this system. Cyanogen bromide peptide profiles of in vivo and in vitro H315 were chromatographically indistinguishable. Three peptides, CN1, CN2, and CN4, which represent approximately 85% of the total amino acids of H315 were isolated and further characterized by electrophoresis and paper chromatography. All were very similar to the corresponding peptides of authentic H315. We conclude that the fidelity of H315 translation is preserved in vitro.


An initiation
study of mineral oil-induced plasmacytoma (MOPC) 315 heavy chain immunoglobulin (H"'") in vitro has been conducted using formyl-[YS]methionyl-tRNA,\"' and a highly purified 18 S message from MOPC 315 solid tumor in a crude rabbit reticulocyte lysate system. The product was specifically precipitated by antibodies directed against MOPC 315 immunoglobulin and H"'". The in vitro H"": products terminally labeled with formyl-[YSlmethionine or internally labeled with ["Hlleucine were electrophoretically identical with in uivo H31s on sodium dodecyl sulfate-polyacrylamide gels. All of the [""SImethionine was incorporated at the NH, terminus, not internally, since there is a near complete recovery of [""S]methionine following one cycle of Edman degradation. The NH,-terminal cyanogen bromide peptide, CN,, of in viuo and in vitro H"" co-migrated exactly on gel electrophoresis under conditions which completely resolved two proteins differing in size by only 14 amino acids. These data strongly suggest that there is no NH,-terminal precursor of H"'j in this system.
Cyanogen bromide peptide profiles of in uiuo and in vitro H"'j were chromatographically indistinguishable. Three peptides, CN,, CN,, and CN,, which represent approximately 85% of the total amino acids of HJ1" were isolated and further characterized by electrophoresis and paper chromatography.
All were very similar to the corresponding peptides of authentic H"'". We conclude that the fidelity of H3*j translation is preserved in vitro.
The cell-free translation of partially purified immunoglobulin heavy chain mRNA has been reported by several laboratories (l-4).
The putative heavy chains synthesized in these systems were found to be antigenically related to authentic heavy chain (2)(3)(4)

RESULTS
Isolation of mRNA -Microsomal RNA was isolated from solid tumor and fractionated on sucrose gradients as described. Nine grams of tumor yielded approximately 22.2 mg of RNA of which 6.6 mg sedimen_ted at 16 to 24 S. Oligo(dT)-cellulose bound 3 to 4% of this fraction on the first purification and 60 to 75% on the second purification, giving a total yield of 100 to 200 pg of mRNA. Fig. 1 shows the purity of our mRNA as analyzed on SDSpolyacrylamide gels. After one purification on oligo(dT)-cellulose the major band migrated with 18 S ribosomal RNA, and five minor bands were visible in the region between the 18 and 28 S markers. A second purification on oligo(dT)-cellulose did not alter the banding pattern.
Cell-free Translation of IgA315 mRNA -Microsomal RNA isolated from solid tumor was fractionated on a sucrose gradient and the RNA sedimenting at 16 to 24 S was translated in a crude reticulocyte lysate. As shown in Table I, there was a high level of protein synthesis in lysates without added RNA. This was due to globin synthesis directed by endogenous message. The immunoassay, however, was very specific. In controls only 0.09 to 0.13% of total radioactivity in protein was precipitated by anti-IgA315. The majority of the RNA that sedimented within the size range of 16 to 24 S is, of course, the 18 S ribosomal RNA. Therefore, even at an input RNA of 800 pg/ml, no translation of heavy chain was detected by anti-IgA315. A small percentage of the RNA was selectively purified by binding to oligo(dT)-cellulose.
The purified RNA directed the translation of significant amounts of antibody-precipitable protein (Table I  We also found that the translation product of 16 to 24 S (dT)retained RNA could be specifically precipitated by anti-H3i5. However, for reasons which are not readily apparent, precipitation with anti-H315 was less efficient and was therefore not routinely used.
Polyacrylamide Gel Electrophoresis of Translation Product-H315 labeled in vitro with 13H11eucine or formyl-[35S]methionine was compared with nonradioactive authentic H315 by polyacrylamide gel electrophoresis (Fig. 2). Native H315 moved as a diffuse band with a concentrated region at the front (Fig. W). The migration of totally reduced and carboxymethylated H3i" was retarded relative to native H315 (Fig. 2F), presumably due to chemical modification.
Since the in vitro H315 was not reduced and carboxymethylated, it can only properly be compared with native H315 (Fig. 2E).
The translation product of H3*" mRNA migrated in four sharp bands with identical R,. values for products labeled with either 13H11eucine or formyl-13S1methionine.
The primary band represented approximately 85% of the total cell-free products and migrated identically with authentic H3*" (RF = 0.390 for cell-free product and 0.393 for native H315). The second band (approximately l,O% of the total) moved slightly ahead of native H31" (R, = 0.408). Two other products (representing less than 5% of the total radioactivity) migrated with Rb. values of 0.502 and 0.605. In the [35S1methionine-labeled sample ( Fig.  20) a faint radioactive band was also present which migrated slightly behind L315 (R, = 0.798 for cell-free product and 0.858 for authentic L31"). This band probably represents the synthesis of a small amount of light chain precursor.
Very faint radioactive bands were also detected in both the control (no RNA) and experimental (H315 mRNA) samples at the dye front and at the rear boundary of authentic H3'". These bands most likely represent nonspecific background radioactivity. [Yj'lmethionine -After deformylation, an Edman degradation was carried out for two cycles on the in vitro H31s to establish that the [35S]methionine was indeed at the NH, terminus. At each cycle the PTH-amino acid derivative was extracted into the ethyl acetate phase. Any contaminating peptide accidentally extracted with butyl chloride should remain in the HCl phase. Table II shows that 82% of the radioactivity was recovered as PTH-amino acid derivative in the first cycle and 6% in the second cycle. Less than 3% of the radioactivity remained in the peptide after two cycles of the Edman procedure. These results confirm that the 13YS]methionine was incorporated only at the NH, terminus.

Comparison of CNBr Fragments of in Vivo and in Vitro
W'" -To further establish the identity of our cell-free H315 with authentic H315, we prepared in vivo [14C]IgA31j as described under "Experimental Procedures." In vitro 13HlH315 and in vivo [14C]IgA315 were combined, totally reduced and alkylated, and chromatographed on Sephadex G-100 in guanidine HCl (Fig. 3). There is a small peak of in vitro protein at Fraction 66 which probably represents dimer. Other than this, the H3'" peaks coincide. The H315 peak was recovered and cleaved with cyanogen bromide (28). Cyanogen bromide cleavage results in six fragments, five of which can be separated. These fragments have been characterized as follows (28). CN, has 228 amino acids; CN, has 156 amino acids and includes the NH, terminus, the entire variable region, and part of the constant region. The weakest and slowest moving band, representing uncleaved H31", moved identically in in vivo and in vitro samples. The band migrating most rapidly represents CN,, and the band following it probably represents a composite of CN, and another CNBr fragment. CN, moved as a diffuse band in both in vivo and in vitro samples, but particularly in the latter. CN, from in vitro H"'" migrated slightly ahead of its in uivo counterpart. However, the more compact band of in viuo CN, moved within the outer boundaries of the in vitro CN, band. As discussed later, this slight difference in the migration of CN, probably reflects a difference in the way in uivo and in vitro samples were handled. Electrophoresis of a mixture of in vivo and in vitro CN, (Fig. 5LI) resulted in one broad band.
The composite fragment from in vitro H31" migrated as a sharp band and had a slightly higher mobility than the corresponding in viuo fragment. A mixture of the fragments from in viuo and in vitro samples (Fig. SD)  NH, terminus, isolated from in uivo and in vitro H"'" is shown in Fig. 6. The position of the band representing CN, from in vitro H31" coincides exactly with that of CN, from authentic H315. The absence of major contaminating composite fragments as seen in the CN, fraction can be largely explained by two facts. Since CN, is well within the separation range of Sephadex G-100 (M, = 16,000) the fragment could be isolated in purer form. Secondly, since CN, is the NH, terminus, only one cleavage is necessary to produce CN, whereas two cleavages are necessary to free CN,, an internal fragment.
Comparison of in Vivo and in Vitro CN, and CN5-The octapeptides, CN, and CN,, were purified on Sephadex G-50 as described under "Experimental Procedures," and aliquots of the in viuo and in vitro samples were electrophoresed adjacent to each other in pyridyl acetate at pH 3.6. The radioactive profiles of in vivo and in vitro samples are identical (Fig. 7). Sharp peaks are evident at the origin and 16 to 17 cm away from the origin toward the cathode. A faint ninhydrin spot is visible at 16 to 17 cm and an even fainter spot at 6 to 8 cm. Based on the amino acid compositions of CN, and CNj,**j we have tentatively identified the peptide at position 16 to 17 as CN,, since it contains a histidine which would be positively charged under these conditions. The ninhydrin spot at 6 to 7  (A), and from in vitro [3HlH31" (B). cm and the small radioactive peak of the in vitro sample in this area may represent CN,.
Aliquots of in vitro and in vivo octapeptides were spotted separately on Whatman No. 3MM paper and subjected to descending paper chromatography.
The results (Fig. 8) show a small peak at the origin and a large peak at 22 to 26 cm in both samples. A single ninhydrin spot marked the location of the peptide from nonradioactive carrier H3'" at the front edge of the radioactive peak (data not shown). The R, of the in vitro peptide (0.657) was virtually identical with that of the in vivo peptide (0.654). When electrophoresed at pH 3.6, the peptide behaved like the peptide identified above as CN,. The small peak at the origin did not move on electrophoresis.

DISCUSSION
The major band of RNA which has been twice purified on oligo(dT)-cellulose and which is active in the synthesis of H3'" migrates with the 18 S marker on SDS-acrylamide-agarose gels (Fig. 1). This is consistent with reports that the mRNAs for MOPC 21  reports that H31j mRNA sediments at 16 to 19 S (3,4) or at 17 S (32, 33) on sucrose gradients.
Our in vitro translation assay was highly reproducible. In a total of 15 experiments, the amount of H3'" synthesized ranged from 0.55 to 1.95% (mean = 1.04%) oftotal protein synthesized. Nonspecific precipitation (background) ranged from 0.04 to 0.21% (mean = 0.12%) of total protein synthesized. Since HaI" represented such a small fraction of total protein, it was imperative that nonspecific precipitation be minimal. We found this assay to be very sensitive and reproducible with various preparations of RNA and of crude reticulocyte lysate.
The protein synthesized in our cell-free system was shown to be related to authentic H31" by several criteria. Our cell-free product had antigenicity for both anti-IgA31Z and anti-I-P'". The size of our primary translation product was identical with that of authentic HaIs as judged by SDS-polyacrylamide gel electrophoresis and gel filtration under denaturing conditions. CN, from native and in vitro H31" co-chromatographed on gel filtration and migrated closely on gel electrophoresis. CNt and CN, moved identically with their authentic counterparts on both electrophoresis and chromatography.
CN, moved identically on Sephadex chromatography but was not analyzed further.
CN, and an unidentified composite fragment from in viuo and in vitro samples differed slightly in mobility on electrophoresis. It is probable that this reflects a slight difference in the treatment of in uivo and in vitro samples. In vivo H3'" was isolated from IgA31s which had been totally reduced and carboxymethylated. In vitro H3'" was not subjected to this treatment. CN, contains 8 cysteine residues3 which were carboxymethylated and could account for slower migration on SDSacrylamide gels. Indeed we have found that after total reduction and carboxymethylation H31J and L31" migrate more slowly on SDS-polyacrylamide gels than H31" and L31s of native IgA3'" which has not been so harshly treated. Fig. 2 shows that native H31" has an RF of 0.393 while totally reduced and carboxymethylated H"'" has an RF of 0.362. Native and totally reduced and carboxymethylated L31" migrate with R,: values of 0.858 and 0.772, respectively (data not shown).
We were unable to precisely locate radioactive CN,, the COOH terminus of H31", in either the in vivo or in vitro sample. Since CN, was labeled only at the third position from the COOH terminus, it is possible that the label was cleaved after synthesis by contaminating carboxypeptidase. Alternatively, it is possible that the specific activity was too low to be detected in the present study. In any case, CN,, CN*, and CN, represent 392 amino acids of HZ'", thus providing excellent evidence for the synthesis of at least 85% of H31".