Polysomes released from microsomes of MOPC 41 mouse myeloma were used to prepare a poly(A)-containing fraction of RNA by chromatography on poly-(dT)-cellulose. From that fraction, a 14S RNA species was purified to a single peak by successive sucrose gradient centrifugations, followed by acrylamide gel electrophoresis. The RNA has an apparent molecular weight of 380,000 (1100 nucleotides), as estimated from the electrophoretic analyses. In a reticulocyte lysate this RNA directs the synthesis of a protein that migrates more slowly in sodium dodecylsulfate-acrylamide gels than does the light chain secreted by the same tumor. This difference in migration corresponds to a size difference appropriate for polypeptide chain about 20 amino acids longer than the light chain. The tryptic peptides of this protein correspond to those of the secreted light chain, except for the presence of two additional peptides from the product synthesized in vitro and for the absence of one light-chain peptide. The purified RNA is, therefore, the mRNA of the light chain, and it seems to code for a precursor protein slightly larger than the light chain. From the estimated size of the 14S mRNA, it appears that only 65% of the RNA is translated.

As previously shown, purified 14S RNA of mouse myeloma MOPC-41 forms a single peak on sucrose gradients and gel electrophoresis and codes for a single polypeptide chain, the immunoglobulin light chain produced by the same myeloma in vivo. This 14S mRNA was used for the enzymatic synthesis of DNA (cDNA) which is complementary to the RNA template. A DNA fraction was isolated which has an average size of 300 nucleotides. A large body of evidence indicates that the variable (V) and the constant (C) regions of immunoglobulin chains are coded by separate V and C genes (1). These two polypeptide sequences, however, are not synthesized separately. The mRNA of light chain can direct the synthesis of complete light chain in heterologous systems (2, 3). Studies on the structure of light chain mRNA indicate that the sequences coding for the V and the C regions are on the same RNA molecule (4).The major theories which have been proposed to explain the generation of diversity of immunoglobulin genes (see ref. 1) allow important predictions concerning the number of immunoglobulin genes in the genome of germ line cells or of various types of somatic cells. The availability of radiolabeled complementary DNA (cDNA), synthesized enzymatically from 14S light chain mRNA and containing sequences complementary to the C gene of immunoglobulins (2, 5), makes possible a kinetic study of its-annealing with unlabeled cellular DNA in large excess (6). This allows a (NIH) and maintained in this laboratory for several years. The preparation of polysomes, extraction of polysomal RNA, fractionation of polysomal RNA by poly(dT)-cellulose chromatography, and isolation-of the 14S mRNA peak coding for MOPC-41 light chain were performed as described earlier (2, 7) with the following modifications: Polysomes at a concentration of 20 A260 units/ml were incubated with Proteinase K (Merck, chromatographically pure) (100 gg/ml)) and Na dodecyl sulfate (0.2%) (KCl, final concentration of 20 mM) for 15 min at 00. The poly(A)-rich RNA was dissolved in H20 (1 mg/ml), heat denatured for 10 min at 650 and chilled immediately to 00. It was then adjusted to 5 mMI Na acetate, pH 5, and 400-,ul aliquots were fractionated on linear 5-20% (w/v) sucrose gradients, containing 5 mM Na acetate, pH 5, in a Spinco SW 41 rotor for 16 hr at 40,000 rpm and 20. The 14S RNA peak was refractionated on a second gradient under identical conditions. Enzymatic synthesis of cDNA from the purified 14S mRNA peak has also been described (5).Preparation of DNA from Liver and MOPC 41 M1yeloma Nuclei. Nuclei were prepared according to Blobel and Potter (8) except that after the first sedimentation, nuclei were treated with the detergent NP-40 (1%) before the centrifugation over a cushion of 2.2 M sucrose. Packed nuclei (3 ml) were suspended in 100 ml of 0.15 M NaCl, 0.015 M Na citrate, pH 7.5, and 19 ml of 10% Na dodecyl sulfate. They were then incubated with Proteinase K (100 Ag/ml) for 4 hr at 45°.Repeated extractions with phenol-chloroform and digestions with RNase were...

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Rauscher spleen focus-forming virus contains functional gag and pol genes and a partially deleted env gene which is structurally related to the env genes of dual tropic murine leukemia viruses. The Rauscher and Friend erythroleukemia viruses contain defective spleen focus-forming virus components (R-and F-SFFVs) which are required for acute pathogenesis in adult mice (9, 27, 28, 38) and for stimulation of erythropoiesis in bone marrow cultures (2, 13). The F-SFFV genome is related to those of replication-competent dual tropic murine leukemia viruses (MuLVs) (35, 36, 38, 39). However, it is substantially smaller and contains partial deletions in its env and pol genes and a small substitution in its gag gene (5, 10). Recently, we reported that R-SFFV encodes a membrane glycoprotein with an apparent Mr of 54,000 (gp54) that is very similar to the env glycoprotein encoded by F-SFFV (28). Furthermore, R-SFFV encodes gag polyproteins which we could not distinguish from those encoded by the Rauscher MuLV helper (28). We now describe evidence that R-SFFV encodes reverse transcriptase and may differ from dual tropic MuLVs only by a partial deletion (approximately 0.7 kilobases [kb]) in its env gene.