Changes in J chain and mu chain RNA expression as a function of B cell differentiation.

Abstract

An immunocompetent B lymphocyte can be triggered by the appropriate antigen and lymphokine signals to differentiate into a pentamer IgM-secreting ceil. The process involves a change in the function of the IgM antibody molecule; it is converted from a membrane receptor for antigen to a secreted polymer that effects antigen disposal. This change is known to require (a) synthesis of the secreted form of # heavy chain and (b) synthesis of the polymerizing component, the Ig J chain.Membrane and secreted forms of the # heavy chain were first distinguished by their different mobilities in polyacrylamide gels (1). Subsequent studies showed that the structural differences are confined to the carboxy-terminal sequence of the chain and correlate with the different functions of the IgM molecule (2, 3). The membrane form has a hydrophobic terminus that anchors the IgM monomer in the B cell membrane whereas the secreted form has a shorter, more hydrophilic terminus that contains the penultimate cysteine residue required for pentamer IgM assembly. The two termini were found to be encoded in exons located 3' to the C# gene and are cotranscribed with the gene (4). Separate ~m and ~ts mRNAs are then generated from the single gene transcript by differential 3' cleavage and processing. These data established that the switch in ~ chain synthesis during B cell differentiation is regulated at the posttranscriptional level.The assembly and secretion of pentamer IgM also depends on the synthesis of a second protein, the J chain (5, 6). The J polypeptide serves to initiate IgM polymerization by bridging two IgM monomers through disulfide bonds to the #s carboxy termini. The resulting J chain-containing dimer then promotes the disulfide bonding of three additional monomers to complete the pentamer structure. Studies of rabbit and human splenocytes (7,8) have shown thatJ chain expression is a direct result of B cell activation. Little or no intracellular J chain could be detected in the unstimulated cell populations. After stimulation, however, large amounts of J chain were observed concomitant with the appearance of secreted IgM in the culture supernatants. Moreover, analyses of J chain mRNA in transformed cell lines showed that J chain-specific sequences were absent from a line representing an unstimulated B cell, but were present in large amounts in a myeloma cell line secreting pentamer IgM (9). These results indicate

Sequences Adjacent to the 5′ Splice Site Control U1A Binding Upstream of the IgM Heavy Chain Secretory Poly(A) Site

“…Briefly, all RNA substrates were first refolded by heating in reaction buffer at 70°C for 2 min followed by cooling to 37°C over a 30-min period. For RNase T1, RNase V1 and lead (II) cleavage, 10,000 cpm of 3Ј-or 5Ј-end-labeled RNA substrate was incubated at 37°C in a total volume of 5 l with either 0.4-0.001 unit of RNase T1 (Roche Applied Science) or 0.072-0.00072 unit of RNase V1 (originally Amersham Biosciences, kind gift of Jean Patterson) in 50 mM PIPES, pH 7.0, 100 mM NaCl, 10 mM MgCl 2 for 5 min or 0.1-0.5 mM Pb(OAc) 2 in 50 mM Tris HCl, pH 7.5, 100 mM, NH 4 Cl, and 10 mM MgCl 2 for 10 min. The reactions were stopped by the addition of 10 M urea, 25 mM EDTA.…”

Accessibility of the promoter sequence in the J-chain gene is regulated by chromatin changes during B-cell differentiation.

“…(14) and a significant number of memory cells that have undergone the early stages of antigen activation (13,27). After mitogen stimulation, however, the PstI site in the J-chain gene became 10-fold more susceptible to cleavage and gave a pattern identical to that found in immunoglobulin-secreting lines.…”

“…Nuclei were isolated from the two cell lines and digested with DNase I at various concentrations. The DNA was then extracted from the nuclei, digested to completion with BamHI, and hybridized in Southern blots with probes [14]). …”

“…The Isolation and culture of normal murine lymphocytes. Single-cell suspensions were prepared from the spleens of 8-to 12-week-old BALB/c mice and size-fractionated by 1 x g velocity sedimentation (14 (38), DNA was isolated from each sample by phenol-chloroform extraction followed by isopropanol precipitation.…”

“…Analyses of J-chain-specific RNA in both mitogen-stimulated B cells and transformed B lymphoma lines have shown that synthesis of the J protein is initiated at the level of transcription (14,18). The J-chain gene remains silent during the early antigen-independent stages of differentiation, but becomes activated when the mature B cell is driven to secretion by contact with antigen and T cellderived lymphokines (24).…”