Opsin, the apoprotein of the visual pigment rhodopsin, is synthesized on membranes of the rough endoplasmic reticulum and subsequently passes through the Golgi apparatus to the rod outer segment. This pathway parallels the early stages of biosynthesis of some secretory proteins and viral membrane glycoproteins. Most of these proteins are initially synthesized as precursor molecules with a short-lived hydrophobic extra peptide segment at the NH2 terminus. Therefore we investigated whether or not the immediate translation product of opsin mRNA contains a similar short-lived NH2-terminal extra peptide. The mRNA coding for opsin was isolated from bovine retina polysomes precipitated by antibodies to opsin. The mRNA directed the cell-free synthesis of a protein comparable in size to opsin that was specifically precipitated by anti-opsin antibodies. Sequence analyses of the immunoprecipitated protein labeled with six radioactive amino acids (Met, Asn, Pro, Phe, Tyr, Val) provided the following result: MATERIALS AND METHODS Anti-Opsin Antibodies. Bovine opsin was isolated electrophoretically from sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gels as described (24). In the presence of 0.1% NaDodSO4 the protein was mixed with an equal volume of complete Freund's adjuvant and injected at 7-day intervals into a goat. Antibody content of sera was tested by precipitin analysis. The initial opsin solution that contained NaDodSO4 inhibited precipitin formation. Therefore the solution of NaDodSO4-solubilized opsin was dialyzed against 1000 vol of 0.15 M NaCl at 20°C. During dialysis a precipitate occasionally formed. The centrifuged supernatant (3000 X g for 10 min) was cooled to 4°C and any additional precipitated material (presumably NaDodSO4 and aggregated protein) was removed by further centrifugation. Over 80% of the opsin was recovered in the clear supernatant, and it formed specific precipitates with anti-opsin antibodies. The dialyzed opsin solution (1 mg/ml) could also be coupled directly to CNBr-activated Sepharose in 0.1 M NaHCO3 (25) Haven, CT 06510. 2654The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci. USA 76 (1979) 2655 135SMet-tRNAMet. The two [35SSMet-tRNAMet species that transfer methionine to the NH2-terminal (initiator MettRNAIN'tt) and internal positio'Siin proteins were prepared from wheat germ as described (27

Critical to our understanding of the immune system diversity is the determination of the number of germ line V genes. The total number of V genes is given by the product: number of subgroups x number of germ line genes per subgroup. Studies of kappa chains and of embryonic DNA indicate 5-10 V genes per subgroup. Statistical analysis of the limited sequence data of mouse kappa chains suggest about 50 V kappa subgroups. We report here a general approach for direct estimation of the number of VL and VH subgroups expressed in normal spleen, and present data for V kappa. The kappa mRNA of the spleen is a heterogeneous population where different V kappa are linked to the same C kappa, i.e. C kappa equals total V kappa. The ratio C kappa/distinct V kappa approximates the number of subgroups since V kappa of the same subgroup cross hybridize while V kappa of different subgroups do not. This ratio was determined by molecular hybridization of cloned C kappa and V kappa DNA probes with spleen mRNA. The results indicate the expression of 280 V kappa subgroups in mouse. Assuming an average of 7 genes per subgroup, we estimate about 2000 V kappa germ line genes.

Early studies have shown that the mRNA molecules coding for mouse immunoglobulin(1g) light(L)-chains direct the cell-free synthesis of precursors somewhat larger than the mature protein, as estimated from electrophoretic mobility in sodium-dodecyl-sulfate(SDS)-polyacrylamide gels.'-' Some understanding of the functions of the precursors may be afforded by determination of their structure, i.e., the position (NH,-or COOH-terminal end), precise size, and primary structure of the extra peptide segment. This was first done in 1973, when the precursor of a mouse Ig L-chain, programmed by mRNA in vitro, was subjected to radioactive sequence analysis.' This study established two features common for all precursor molecules: in the precursor an extra peptide segment (about 20 residues long) precedes the NHz-terminus of the mature protein; this extra piece is enriched with leucine residues, thus indicating the marked hydrophobicity of the extra piece. Subsequently it was found that many other secretory proteins and integral membrane proteins were initially synthesized as precursor molecules having similar short-lived NH2-terminal extra pieces (see references 5 , 6, and this volume).The Ig molecule is composed of two L-chains and two heavy (H)-chains. The L-and H-chains comprise a very heterogeneous population of proteins, and they have unique structural features that make the study of their precursors of special interest. In the mature Ig chain the variable-region (V-region, the 110 NHderminal residues) exhibits sequence variability that is responsible for antibody diversity and specificity; the constant-region (C-region, the COOH-terminal portion of the protein) of H and L chains have a distinct sequence. We have isolated from myeloma tumors of mouse and rat the mRNA molecules that direct the cell-free synthesis of precursors, in which extra pieces (17-29 residues long) precede the NH,-termini of both the V-region (precursors of intact L-or H-chains) and Cx-region (precursor of the retype C-region fragment). The complete sequence of the NH,-terminal extra pieces of 10 Ig precursors were determined (FIGURE 1). The primary structures of these extra pieces and results of other experiments bear on problems of protein secretion in general and interaction of the precursor with cell membranes (endoplasmic reticulum and plasma membrane), as well providing new information concerning the organization and controlled expression of Ig genes.The Ig mRNAs were isolated from mouse or rat myeloma tumors that synthesize discrete Ig molecules. Tumors and their corresponding Ig chains are designated as M-321, MPC-11, IR-102, etc. The mRNAs were prepared from myeloma polysomes specifically precipitated by antibodies' directed to L-or H-chain.',' The Annals New York Academy of Sciences mRNAs were translated in the wheat germ cell-free system to provide precursor molecules that were subjected to radioactive amino acid sequence analy~es. ',~ SEQUENCE VARIABILITY AT THE NH,-TERMINAL EXTRA PIECE OF IMMUNOGLOBULIN PRECURSORS; THE VARIABLE-REGION GE...

We have cloned double stranded cDNA sequences encoding a mouse immunoglobulin light chain (L-321) into the PstI site of the beta-lactamase gene of plasmid pBR322 by the oligo (dG)-oligo (dC) tailing procedure. Escherichia coli X1776 transformed by the recombinant plasmids were screened for the expression of L-321 antigenic determinants by a newly developed in situ radio-immunoassay. One out of seven transformants screened was found to synthesize an L-chain like protein. Each bacterial cell produces about 550 molecules of the L-chain sequence. Preferential segregation of the L-chain sequence to the periplasmic space suggest covalent attachment of the L-chain sequence to the N-terminal portion of beta-lactamase. Restriction mapping of the plasmid DNA isolated from the positive clone indicated the presence of a DNA sequence coding for the entire constant region and extending into the variable region for a length corresponding to about 40 amino acid residues. The orientation of the cloned cDNA with respect to the plasmid DNA is compatible with the formation of a fused beta-lactamase-L-321 peptide.

The mRNA coding for the K-type constant region (C,,) was purified from two clones derived from the MPC-1I mouse myeloma. This mRNA directs the cell-free synthesis of a C, precursor (molecular weight, about 15,000) in which an extra piece, 17 residues long, precedes the NH1ter-minal residue (Ma'19) of the C. region. Te partial sequence of the extra piece is: Met-X-Thr-Asp-Thr-Leu-Leu-Leu-Trp ValLeu-Leu-Leu-Trp-Val-Pro-X-(X is unknown). Met1 was shown to be the initiator methionine. The sequence of the C, extra piece is completely different from any known sequence preceding residue Ma'° § in whole light (L) (i) translocation of this V gene to the C gene, deletion of the entire mature V gene, and "end-toend" repair of the remaining xp-DNA to the C gene; (ii) translocation to the C gene only of the xp-DNA portion of the V gene.