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Immunoglobulin class-switch recombination (CSR) gives rise to looped-out circular DNA of a cleaved S segment, which is lost eventually after cell divisions. To understand the molecular mechanism of S region cleavage during CSR, we constructed artificial CSR substrates in which inversion-type CSR takes place to retain the cleaved S segment. Sequencing analyses of recombinant clones of these substrates revealed that varying degrees of deletions and duplications exist at CSR breakpoints, suggesting the involvement of staggered cleavage of the S region in CSR. In addition, mutations frequently found near junctions showed a similar profile of base replacement to Ig somatic hypermutation. These findings suggest that single-strand tails of staggered cleavage may be repaired by error-prone DNA synthesis.

Signal transduction initiated by crosslinking of antigen-specific receptors on T- and B-lymphoma cells induces apoptosis. In T-lymphoma cells, such crosslinking results in upregulation of the APO-1 ligand, which then interacts with induced or constitutively expressed APO-1, thereby triggering apoptosis. Here we show that crosslinking the membrane immunoglobulin on human lymphoma cells (Daudi) (that constitutively express APO-1) does not induce synthesis of APO-1 ligand. Further, a noncytotoxic fragment of anti-APO-1 antibody that blocks T-cell-receptor-mediated apoptosis in T-lymphoma cells does not block anti-mu-induced apoptosis. Hence, in B-lymphoma cells, apoptosis induced by signaling via membrane IgM is not mediated by the APO-1 ligand.

Somatic mutation of the variable (V) regions of immunoglobulin genes occurs in vivo at rates that have been estimated to be between 10(-3) and 10(-4) per bp per generation. To study this process in vitro, the 18.81 pre-B-cell line and hybrids derived by fusing 18.81 to the NSO myeloma fusion partner were transfected with a mu heavy-chain construct containing a nonsense mutation in the V region (Vn) or the constant region (Cn). Mutation was quantitated by reversion analysis using the ELISA spot assay to detect single cells secreting IgM. Fluctuation analysis revealed that V-region mutations spontaneously occurred in 18.81 cells at an average rate of 5.8 x 10(-6) per bp per cell generation and in selected 18.81-NSO hybrids at greatly increased rates of 1.6 x 10(-3) to 5.8 x 10(-4) per bp per generation. The Vn construct also reverted frequently in transgenic mice, indicating that it contained sufficient information to mutate at high rates both in vivo and in vitro. Sequence analysis of reverted genes revealed that reversion was due to point mutations. Since the rates and nature of the mutations that are occurring in these transfected genes are similar to those reported in vivo, it should be possible to use this system to identify the cis-acting sequences and trans-acting factors that are responsible for V-region somatic hypermutation.

We describe a system to evaluate the function of lymphocyte-specific and generally expressed genes in the differentiation and/or function of lymphocytes. RAG-2 (recombination-activating gene 2)-deficient mice have no mature B and T lymphocytes due to the inability to initiate VDJ recombination. Blastocysts from RAG-2-deficient mice generate animals with no mature B and T cells following implantation into foster mothers. However, injection of normal ES cells into RAG-2-deficient blastocysts leads to the generation of somatic chimeras with mature B and T cells all of which derive from the injected ES cells (referred to as RAG-2-deficient blastocyst complementation). Complementation of RAG-2-deficient blastocysts with mutant ES cells heterozygous for a targeted mutation that deletes all immunoglobulin heavy-chain joining (JH) gene segments (JH+/-) also leads to generation of chimeras with normal B and T cells. However, complementation with ES cells homozygous for the JH mutation (JH-/-) generates animals with normal T cells but no B cells, due to a block in B-cell development at a very early stage. Transfection of a functionally assembled mu heavy-chain gene into the JH-/- ES cells prior to blastocyst injection rescues the JH-/- mutation and allows the generation of both mature T and mature B cells. The rescued B cells express IgM but not IgD and respond normally to bacterial lipopolysaccharide stimulation by proliferating and by secreting IgM.

In several animal viruses, enhancers have been implicated in both DNA replication and transcriptional activation. The linkage of the two mechanisms appears intimate, in that common DNA binding factors can be shared. The immunoglobulin heavy chain (Igh) intronic [heavy chain joining region (JH)-mu chain constant region (C mu)] enhancer (E mu) is required for tissue-specific transcription of Igh genes and is essential for somatic recombination of diversity (D) and J segments. We show here that E mu is located at or near an origin of chromosomal DNA replication, which is more active in B lymphocytes than fibroblasts. E mu does not fulfill two criteria demonstrated for some cellular origins. E mu can initiate but not maintain autonomous replicating activity in B cells. E mu is unable to impart early replication timing to a transfected VDJ-C mu Igh locus in B cells. Instead we propose that E mu-associated ori activity contributes to tissue-specific Igh expression through local effects on chromatin structure leading to subsequent accessibility of transcription and/or recombination factors for the enhancer.

A 90-kDa phosphoprotein (p90) of the endoplasmic reticulum was identified by a monoclonal antibody generated against human hepatoma cells. Pulse-chase experiments with [32P]phosphate and [35S]methionine demonstrated that p90 formed both stable and transient complexes with other cellular proteins, suggesting its role as a molecular chaperone. This protein associates with heavy chains of major histocompatibility complex class I proteins, suggesting that it is the human homolog of the recently described 88-kDa protein that transiently associates with murine class I molecules in the endoplasmic reticulum. The p90 protein also associates in B lymphocytes with membrane immunoglobulin mu heavy chains and may serve as a chaperone for many membrane-bound polypeptides. A partial human p90 cDNA was cloned from a lambda gt11 expression library and identified as the human homolog of calnexin, a major canine calcium-binding protein found to be associated with the signal-sequence receptor in endoplasmic reticulum membranes.

B lymphocytes expressing surface IgM with or without IgD may switch to the expression of other isotypes (IgG, IgA, or IgE) in the course of immune responses. Analyses of genomic DNA from cloned myelomas and hybridomas have shown that the isotype switch is accompanied by a rearrangement characterized by deletion of DNA between the switch (S) region of the mu gene and that associated with the new isotype, resulting in the formation of a composite S region. Measurement of this deletional rearrangement has been difficult in populations of normal B cells but would be useful for investigating the mechanism of the rearrangement and determining whether deletional rearrangement is responsible for all instances of class switching. We have developed a sensitive assay for deletional rearrangement that we designate the digestion-circularization polymerase chain reaction (PCR). In this assay, genomic DNA is digested with a restriction enzyme that recognizes sites that flank the recombined composite S region. The digested DNA is then ligated at low concentrations to favor the formation of circles. The ligation joins the 5' and 3' ends of each restriction fragment, making it possible to amplify by PCR across the ligated restriction site by using appropriate primers. From DNA that has undergone deletional rearrangement, a single-sized PCR product is produced and can be quantitated. We demonstrate here that the digestion-circularization PCR assay can detect S mu-S gamma 1 rearrangements in B cells cultured with lipopolysaccharide and interleukin 4. The assay is sensitive enough to quantitate switched cells constituting only 1-2% of the population.

The B-cell antigen receptors consist of membrane immunoglobulins (mIgs) noncovalently associated with two accessory proteins, MB-1 and Ig-beta. We used transfection into a nonlymphoid cell line to test whether MB-1 and Ig-beta were sufficient to promote cell surface expression of mIgM capable of signal transduction. Expression of MB-1 and Ig-beta, but not MB-1 alone, allowed high-level surface expression of mIgM in the AtT20 endocrine cell line, which presumably lacks other B-cell-specific components. The reconstituted antigen receptor was capable of mediating some of the signaling reactions characteristic of mIgM in B lymphocytes. Crosslinking mIgM on transfected AtT20 cells stimulated tyrosine phosphorylation of MB-1 and Ig-beta and also increased the amount of phosphatidylinositol 3-kinase activity that could be precipitated with anti-phosphotyrosine antibodies. When total cell lysates were analyzed by anti-phosphotyrosine immunoblotting, however, no induced phosphorylation of more abundant proteins was detected. Moreover, crosslinking of the receptor in AtT20 cells did not stimulate inositol phospholipid breakdown. Thus, the transfected B-cell antigen receptor could initiate some signal transduction events but AtT20 cells may lack components required for other signaling events associated with mIgM.

Wild-type p53 protein is a growth modulator whose inactivation has been found to be a key event in malignant transformation. Reconstitution of wild-type p53 in the p53-nonproducer, Abelson murine leukemia virus-transformed pre-B-cell line L12 gave rise to stably growing clones. Wild-type p53-producer derived cell lines exhibit an altered cell cycle, however. More cells with an extended G0/G1 phase were found than in the p53-nonproducer parental cell line. Furthermore, when injected into syngeneic mice, these cells induced a lower incidence of tumors and these tumors were less aggressive. Analysis of immunoglobulin expression revealed that wild-type p53 induced the expression of cytoplasmic immunoglobulin mu heavy chain. In addition, these derived cells lines exhibited increased levels of a B-cell-specific surface marker, B220. These results suggest that wild-type p53 may function as a cell differentiation factor that can induce development of pre-B cells into a more advanced stage in the pathway of B-cell maturation. In these pre-B cells, wild-type p53 may induce cell differentiation without terminal growth arrest of the cell population.

Precursors of B cells, which constitute a subpopulation of the lymphocytes in bone marrow, can be identified by their surface expression of nonimmunoglobulin markers and the absence of immunoglobulin kappa and lambda light chains. Most pre-B cells synthesize mu heavy chains but, without light-chain partners, these undergo rapid cytoplasmic degradation. In the present study, we demonstrate that late stage pre-B cells, like their neoplastic counterparts, express low levels of a surface receptor composed of mu chains paired with a surrogate light-chain complex formed by Vpre-B and lambda 5-like proteins. The data define a previously suspected but unrecognized stage in normal pre-B-cell differentiation. Expression of a clonally diverse receptor renders this population of immature B-lineage cells potentially vulnerable to clonal selection by antigens and idiotypic interactions.

The universal rule of TA/CG deficiency-TG/CT excess endures the extremely high mutation rate of a retrovirus (human immunodeficiency virus type 1) as well as methylation of CAG rather than CG in a plant (maize). Among the consistently abundant nucleotide oligomers, there are two complementary pairs of palindromic nucleotide pentamers containing TG and CA. Out of the CAGTG and CACTG pair emerged the heptameric pair for the long-distance recombination of immunoglobulin genes, CACAGTG and CACTGTG. Reflecting their origin, these heptamers are found everywhere in all DNA, and a substantial fraction of them are accompanied by nonameric components properly spaced from them. It appears that, were the recombination event not confined to B cells, results of illegitimate recombinations might be disastrous. The other pentameric pair is TGCAT and ATGCA. Out of this pair emerged the complementary pair of transcription enhancer decamers: TNATTTGCAT for immunoglobulin light chains and ATGCAAATNA for immunoglobulin heavy chains. Again reflecting their origin, these decamers are found everywhere in all DNA and some genes--for example, in the 3' flanking region of immunoglobulin heavy chain constant region--are accompanied by a downstream "TATA box." It seems that even with regard to the productively recombined immunoglobulin genes, misinitiation of enhanced transcription is a real possibility.

We have used cloned nucleotide sequences as probes to search for immunoglobulin RNA in T and B lymphoid and nonlymphoid tumor cell lines. Polyadenylated RNA from the cells was fractionated electrophoretically by size and fixed to diazobenzyloxymethyl-paper, and then immunoglobulin RNA species were revealed by hybridization with 32P-labeled cloned sequences for mu heavy chain and kappa light chain. kappa mRNA was detected only in B lymphoid lines known to synthesize kappa chains. No mu RNA was detectable in erythroleukemia, mastocytoma, or sarcoma cells. RNA bearing mu constant region (C mu) sequences, however, was detected in four of nine T lymphoma and four of five myeloid tumor cell lines tested, as well as in B lymphoma and "pre-B" Abelson lymphoma cells. In contrast to the single mu RNA species of 2.6 kilobases (kb) in a plasmacytoma, individual T lymphoma and myeloid lines yielded up to three discrete mu RNA species of apparent size 1.9, 2.2, and 3.0 kb, each different from the two mu RNA species in a B lymphoma line (2.4 and 2.7 kb) or the single species in "pre-B" lymphoma cells (2.9 kb). Both chromosomal complements of the C mu gene in STRij-4 T cells were found to be rearranged from their embryonic (germ line) context. The results suggest that the C mu gene functions not only in T cells, wherein the ability to recognize antigen is long established, but also in myeloid cells.

The mu-chain gene is expressed differently in successive stages of B-lymphocyte development. The heavy chain product appears as a cytoplasmic constituent in pre-B-cells, as part of the IgM receptor in maturing B cells, and as a component in the pentamer IgM antibody synthesized and secreted by the antigen-stimulated cell. We have used the methylation of CpG sequences as an assay system to define the chromatin changes associated with different expression of the mu-chain. The methylation status of eight index sites was followed by restriction enzyme analysis of murine cell lines representing the major stages in the developmental pathway. The analyses showed that a single Msp I/Hpa II site 5' to the immunoglobulin enhancer becomes undermethylated with the onset of mu-chain gene transcription. Four midgene Msp I/Hpa II sites exhibit a progressive loss of methyl groups unrelated to changes in mu-chain gene expression, whereas a Msp I/Hpa II site and two Hha I sites surrounding the exon encoding the carboxyl terminus of the secreted form of mu chain (mus) become undermethylated during the transition to IgM secretion. These results indicate that structural changes in local regions of the mu-chain gene correlate with specific developmental events.

Purified mRNAs coding for mu and kappa human immunoglobulin polypeptides were translated in vitro and their products were characterized. The mu-specific mRNAs, derived from both human lymphoblastoid cells (GM607) and from a mouse-human somatic cell hybrid secreting human mu chains (alpha D5-H11-BC11), were copied into cDNAs and inserted into the plasmid pBR322. Several recombinant cDNAs that were obtained were identified by a combination of colony hybridization with labeled probes, in vitro translation of plasmid-selected mu mRNAs, and DNA nucleotide sequence determination. One recombinant DNA, for which the sequence has been partially determined, contains the codons for part of the C3 constant region domain through the carboxy-terminal piece (155 amino acids total) as well as the entire 3' noncoding sequence up to the poly(A) site of the human mu mRNA. The sequence A-A-U-A-A occurs 12 nucleotides prior to the poly(A) addition site in the human mu mRNA. Considerable sequence homology is observed in the mouse and human mu mRNA 3' coding and noncoding sequences.

Prolonged treatment of mice, starting at birth, with rabbit anti-mouse mu-chain antibodies resulted in the elimination of immunoglobulin-bearing B cells in these animals. The ability of these animals to elicit antigen-specific delayed-type hypersensitivity or cytotoxic T-cell responses to azobenzenearsonate-coupled spleen cells was not impaired. The effect of anti-mu treatment on the restriction by immunoglobulin heavy-chain genes (Igh) of suppressor T cells was investigated. We found that first-order suppressor T-cell factor ( TsF1 ) obtained from anti-mu treated animals expresses an Igh restriction pattern distinct from that observed with TsF1 from normal untreated mice. Furthermore, TsF1 prepared from anti-mu treated animals did not express the major crossreactive idiotypic determinants normally present in TsF1 . The significance of these findings in relation to the role of immunoglobulin on the T-cell repertoire is discussed.

We have used cloned mouse and human heavy chain genes to identify regions of conserved homology between mouse and man and between mu and alpha genes. Using heteroduplex mapping, we find that coding segments for homologous domains appear to have been well conserved between the two species, but the short intervening sequences that separate domains have diverged considerably. These studies also identify extensive regions of homology 5' to both mu and alpha constant region genes that are conserved between the two genes as well as between mouse and man. These segments encompass regions in which mu/alpha recombination occurs during the heavy chain class switch, and their extensive homology may be relevant to this process.

We have produced cell hybrids between mouse myeloma cells, which do not produce immunoglobulin chains, and Burkitt lymphoma cells (Daudi), which express surface IgM. Daudi Cells carry a reciprocal chromosome translocation between chromosomes 8 and 14, described as (8;14)(q24;q32). The hybrids were studied for the expression of human immunoglobulin chains and human isozyme markers, for the presence of human chromosomes, and for the presence of the human genes for heavy chain variable regions (VH) and mu and gamma chain constant (C) regions. The results indicate that the expressed mu chain gene is on normal chromosome 14 in Daudi cells. We have also determined that the chromosome 14 involved in the translocation (14q+) carries the gene for C mu and C gamma 1-4 and probably several genes for the variable region (V). Certain hybrids had lost both the chromosomes 14 but had retained the abnormal chromosome 8 (8q-) that carries the terminal end of the long arm of chromosome 14. These hybrids were studied for the presence of human VH, C mu,, and C gamma DNA sequences, and the results indicated that the hybrid cells with the 8q- chromosome contained VH genes that not C genes. Therefore, we conclude that, in the Daudi Burkitt lymphoma, the break in chromosome 14 occurred within the chromosome segment containing V region genes. As a result of the translocation some of these VH genes became associated with chromosome 8. It is possible that the expression of malignancy in Burkitt lymphoma is caused by immunoglobulin V region gene translocation resulting in activation of a gene on the long arm of human chromosome 8.

The clonal anergy theory of induction of immunological tolerance states that differentiating B lymphocytes that encounter multivalent antigen at the pre-B to B cell transition stage can receive and store a negative signal, which renders them anergic to later triggering stimuli. The theory was tested by using an anti-mu chain monoclonal antibody, E4, as a model tolerogen. The fluorescence-activated cell sorter was used to select B cell-free cell populations from adult murine bone marrow or newborn spleen, and later, to analyze B cell neogenesis in vitro. The presence of E4 at greater than or equal to 1 microgram/ml was required to impede the development of normal numbers of B cells with full receptor status. The subsequent capacity of these B cells to respond in vitro to mitogens was assessed in a filter-cell free microculture system that allows single B cells to proliferate and differentiate. Concentrations of E4 far below those required to affect B cell neogenesis had profound inhibitory effects on the subsequent functional capacity of the B cells. In fact, 10(-3) micrograms/ml of E4 markedly impaired both proliferation and antibody formation, and 10(-1) micrograms/ml, which had no effect on Ig receptor development, abrogated functional capacity. Thus B cells formed in the presence of E4 at 10(-1) micrograms/ml, though possessing the receptor status typical of B cells, were functionally entirely anergic. Exposure to E4 appeared to accelerate the spontaneous death rate of newly formed B cells in vitro. Whether the anergic cell would also have a shortened life span in vivo is not known.

We report here the occurrence of homologous recombination between transferred and chromosomal immunoglobulin genes. Specifically, we have corrected a chromosomal immunoglobulin gene mutation by transferring pSV2neo vectors encoding the constant region of the immunoglobulin mu heavy chain to mutant hybridoma cells that bear a 2-base-pair deletion in the third constant region exon of their chromosomal mu gene. After DNA transfer, we detected G418-resistant transformants that produce normal IgM. Analysis of the DNA structure of the mu gene in these transformants indicates that in four of five cases the mu gene has been restored as a result of the integration of a single copy of the transfer vector by a reciprocal homologous recombination event; the fifth case seems to have resulted from gene conversion or double crossover. These results suggest that this technology might be adapted for mapping immunoglobulin gene mutations by marker rescue and for more convenient engineering of specifically altered immunoglobulin.

Bacterial lipopolysaccharide (LPS) induces proliferation of resting primary murine B lymphocytes and their differentiation into Ig-secreting cells. This is accompanied by an increase in the rate of Ig gene transcription and the accumulation of mu heavy chain secretory mRNA. Specific antiantigen receptor antibody (anti-mu) induces resting B cells to proliferation but not differentiation. Upon addition of both LPS and anti-mu to cultures, resting B cells again proliferate but do not differentiate. RNA transfer blots of the Ig mRNA 2 days after induction with LPS/anti-mu show a specific deficiency of the 2.4-kilobase (kb) mu secretory mRNA, whereas the levels of the 2.7-kb mu membrane and 1.2-kb kappa light chain mRNAs are as high as in cells treated with LPS alone. Between days 3 and 4 after treatment with both reagents, reductions of mu membrane and, to a smaller extent, kappa mRNA become apparent. As measured by nuclear run-on transcription experiments at day 2, the transcription rates of Ig mu and the Ig kappa transcription units are equal in both induction experiments. Only at later stages do the LPS/anti-mu-treated cells transcribe Ig genes at a lower rate. Thus, the anti-mu treatment, drastically reducing the mu secretory mRNA production at early stages, represents a negative regulation occurring primarily at the posttranscriptional level.

We describe here a murine Ly-1-bearing pre-B-cell tumor that, when induced for kappa light chain expression with bacterial lipopolysaccharide, also gives rise spontaneously to a few percent of cells expressing surface lambda light chains. These lambda-positive cells have undergone DNA rearrangements involving either V lambda 1 or V lambda 2 genes. Nearly all clones of lambda-bearing cells express mu and lambda on their surface (but not kappa). However, all these lambda-positive clones continue to transcribe kappa mRNA and synthesize internal kappa chains. Further, surface lambda-positive clones show JH rearrangements on one or both heavy chain chromosomes.

mRNAs encoding the membrane-associated (mu m) and secreted (mu s) forms of mu heavy chain are derived from transcripts of the same immunoglobulin gene by differential RNA processing. To help elucidate the mechanism that regulates the production of these two mu mRNAs during the course of B-lymphoid maturation, we produced a series of specifically modified mu-chain genes and studied their expression when transfected into cells representing either early or late developmental stages. We have established that proper regulation depends on linkage of the mu s and mu m poly(A) addition sites and the length of the mu s-mu m intron. Deletion of an 800 to 900-nucleotide segment from the central region of this intron abolishes regulation; replacement of this segment with miscellaneous DNA sequences restores it. From these results we propose a model in which regulation is principally achieved by competition between cleavage/polyadenylylation of the mu s site and splicing of the C mu 4 and mu m exons.

The complete amino acid sequence of th variable (V) region of the mu heavy chain of a human IgM immunoglobulin (Cam) has been determined. The strategy for sequence determination involved sequenator analysis of the CNBr cleavage products of the succinylated carboxymethylated Fab mu fragment, and of tryptic peptides of the CNBr polypeptides and thermolytic subpeptides. The variable region of this heavy chain (VH) belongs to the VHIII subgroup; it has greater than 70% homology with other VHIII sequences and contains the VHIII marker peptide, Phe-Thr-Ile-Ser-Arg (residues 67-71). As more sequences have been published, the number of subgroup-specific residues has diminished to the point that no position is absolutely subgroup specific. An analysis of the available human VH sequences in the V/C switch region showed the likelihood of a human JH segment (residues 101-113) analogous to the J segments in mouse light chains. The JH region is highly conserved, has striking homology to proposed mouse JH regions, and has significant homology to known mouse J lambda and J kappa segments.

Human pre-B cells, which produce immunoglobulin heavy chain but do not produce immunoglobulin light chain, are shown to contain a 1-kilobase transcript homologous to immunoglobulin lambda light-chain genes. Detailed analysis of RNA and cDNA clones derived from these transcripts reveals that they originate from the distinct immunoglobulin lambda-like genes 14.1/16.1. Sequence analysis of these clones reveals a long open reading frame, beginning with an ATG, capable of encoding a protein of 214 amino acids with an unprocessed molecular weight of 22,944. The C-terminal half of this predicted protein is highly homologous to immunoglobulin lambda light-chain joining and constant region protein sequence, while the amino-terminal end does not share homology with variable regions. Unlike immunoglobulin genes, these genes do not undergo rearrangement prior to expression. Analysis of a panel of 26 hematopoietic cell lines reveals that expression of 14.1/16.1 is limited to pre-B cells and one B-cell line, which, like the pre-B cells, is surface immunoglobulin negative. Antisera raised against a peptide whose sequence was predicted from the 14.1 cDNA sequence identifies a 22-kDa protein in human pre-B cells. Immunoprecipitation of immunoglobulin mu-chain from these pre-B cells with anti-immunoglobulin mu antibody coprecipitates a 22-kDa protein, which is a candidate for the human immunoglobulin omega light-chain protein and may be the protein product of the 14.1/16.1 genes.

The complete amino acid sequences of the variable regions of two human IgM anti-gamma globulins are reported. The two proteins, Sie and Wol, share idiotypic determinants that are distinct from those shared by the two previously sequenced IgM anti-gamma globulins, Lay and Pom [Capra, J. D. & Kehoe, J. M. (1974) Proc. Natl. Acad. Sci. USA 71, 4032-4036; Klapper, D. G. & Capra, J. D. (1976) Ann. Immunol. (Inst. Pasteur) 127C, 261-271.] In contrast to Lay and Pom, Sie and Wol have very little homology in the complementarity-determining hypervariable regions of either their light or heavy polypeptide chains. However, there are only two amino acid differences in the framework portions of the light chains, and the J segments of both chains are homologous. These data suggest that the predominant idiotypic determinant in this cross-idiotypic system may be different than in the Po group and may be either related to their light chain framework structures or to the amino acid sequence in the J segments of these molecules.

The retroviral oncogene v-rel transforms poorly characterized lymphoid cells. We have explored the nature of these cells by analyzing the configuration and expression of immunoglobulin genes in chicken hemopoietic cells transformed by v-rel. None of the transformed cells expressed their immunoglobulin genes. The cells fell into three classes: class I cells have their immunoglobulin genes potentially in an embryonic configuration; class II and class III cells have lost one copy of the lambda light chain locus and have one copy of the heavy chain locus rearranged into a configuration that differs from what is found in mature B cells. In class II cells, the other heavy chain locus may be in embryonic configuration, whereas it is deleted in class III cells. The first of these classes may represent the earliest stage of the lymphoid lineage yet encountered among virus-transformed cells, whereas the second and third classes represent an apparently anomalous rearrangement whose origin remains unknown.

We have studied immunoglobulin double-isotype expression in a transgenic mouse (TG.SA) in which expression of the endogenous immunoglobulin heavy chain locus is almost completely excluded by a nonallelic rearranged human mu transgene. By flow-cytometric analyses, we have shown that a small, but significant, portion (about 4%) of transgenic spleen cells expresses human mu (transgene) and mouse gamma (endogenous) chains when cultured in vitro with bacterial lipopolysaccharide and interleukin 4. By using amplification of cDNA by the polymerase chain reaction, followed by cloning and sequencing of the amplified cDNA fragment, we have demonstrated expression of trans-mRNA consisting of the transgenic variable and endogenous constant (gamma 1) region sequences. Such trans-mRNA could be produced by either switch recombination or trans-splicing between the transgene and endogenous sterile gamma 1-gene transcripts. These results indicate that trans-splicing might be a possible mechanism for the immunoglobulin double-isotype expression in normal B lymphocytes that have not rearranged the second expressed constant region gene.

Considerable evidence has accumulated suggesting that the antigen receptor(s) on T cells is coded for by genes for the variable (V) region of the immunoglobulin heavy (H) chains. In B cells, a complete gene for the immunoglobulin VH region is formed by somatic recombination of VH and joining region heavy chain (JH) gene segments [through an intermediate diversity(D) region gene segment]. In an attempt to determine whether a complete immunoglobulin VH region is expressed on T cells that bear an antigen receptor, we analyzed the restriction map of the JH-C mu locus in genomic DNA from two cloned murine cytotoxic T-lymphocyte (CTL) lines specific for the x-ray-induced leukemia RL male 1. We found no rearrangement of the JH C mu locus in the CTL lines, indicating that the T-cell antigen receptor(s) in these CTLs is not coded for by a complete immunoglobulin VH gene formed by joining of VH, (DH), and JH genes. In addition, we determined that C mu genes on both chromosomes were present and that there was no rearrangement of the C alpha, C kappa, or lambda chain genes in these CTL cells.

The cloned murine B-cell lymphoma line (BCL1) that expresses surface IgM and IgD is considered to be a model for the immunoglobulin gene expression of the mature virgin B cell. Of particular interest is the mechanism by which a single VH gene is shared by two CH genes. We examined the organization of the immunoglobulin heavy chain genes in BCL1 DNA. A single arrangement of CH genes was found with the expressed VHDJH gene complex just 5' to the Cmu gene. The complete DNA sequence of the VH gene was determined. No rearrangement occurred in the intervening DNA between the JH and C mu genes or between the C mu and C delta genes. We conclude that dual expression of mu and delta heavy chains using a single VH gene is accomplished by alternate processing of a primary transcript that encompasses the the VHDJH complex and both CH genes.

The gel filtration behavior, in the presence of detergents, of membrane-bound IgM from normal mouse spleen B lymphocytes was compared to that of secretory IgM from mouse plasma cells. The proteins were labeled either by surface radioiodination or biosynthetically with radioactive amino acids. Cell lysates were fractionated on calibrated Sepharose 6B columns in the presence of the detergents Nonidet P-40 or deoxycholate. Eluted fractions were immunoprecipitated and the reduced or unreduced precipitates were analyzed by sodium dodecyl sulfate gel electrophoresis followed by radioautography. Surface 125 I-labeled 8S IgM exhibited a gel filtration pattern in Nonidet P-40 corresponding to much higher apparent molecular weight than that of secretory 8S IgM, a difference that almost disappeared when gel filtration was performed in the presence of deoxycholate, which forms much smaller micelles than does Nonidet P-40. Biosynthetically labeled lymphocytes contain two types of IgM molecules differing in their gel filtration behavior and fate: one identical to secretory 8S IgM of plasma cells and secreted in the medium during chase periods, and the other identical to surface 125 I-labeled IgM and remaining cell-associated. Because the surface-bound 8S IgM was not found to be associated with other labeled molecules, it is likely that the detergent-binding behavior of surface IgM is due to a hydrophobic segment carried by these Ig molecules. That lymphocytes synthesize two types of μ chains was also shown by the use of tunicamycin, an inhibitor of glycosylation. In its presence, two unglycosylated μ chains were observed: one identical in size to that made by tunicamycin-treated plasma cells, and the second slightly larger. Gel filtration in Nonidet P-40 of the cell lysates of tunicamycin-treated lymphocytes showed that the nonsecretory 8S IgM contains this second type of μ chains, whereas the IgM molecules of the secretory type contain plasma cell-like μ chains. It is suggested that membrane IgM μ chains contain a hydrophobic segment which is responsible for its association to the membrane.

Six independently maintained sublines of FLEB 14, a fetal-liver-derived Epstein-Barr virus-transformed pro-B cell line that has not yet rearranged its immunoglobulin genes, were examined after in vitro propagation during 19-36 months. Two lines showed no immunoglobulin heavy chain gene rearrangement, whereas one allele was rearranged with breakpoints inside the switch region of the mu chain gene in the remaining four. These rearrangements had been generated by the translocation of different chromosome fragments to the immunoglobulin heavy chain gene cluster-carrying 14q32 band in each of the four lines. Previously, a similar rearrangement was found in a fifth subline concurrently with a reciprocal 6;14 translocation. The transposed pieces have been derived from chromosomes 16 and 18 in two of the more recently rearranged lines. Their origins could not be determined in the remaining two lines, but they were different from each other and the other three 14q+ markers. The 14q+ marker-carrying variant has replaced its diploid progenitor suggesting that the translocation has conveyed some in vitro growth advantage on its carrier. This was also supported by the duplication of the 14q+ marker and the loss of its normal chromosome 14 homologue in one subline during serial culturing. The vulnerability of the switch region of the mu chain gene to illegitimate recombination at the pro-B stage and the possible relevance of this finding for the origin of the Burkitt lymphoma-associated 8;14 (immunoglobulin heavy chain gene cluster/MYC) translocation is discussed.

We have cloned an active gene for an immunoglobulin mu heavy (H) chain, bearing the variable (VH), joining (JH), and constant (C mu) sequences expressed in the IgM-secreting mouse plasmacytoma HPC-76. The mu gene was formed by somatic recombination between a VH gene and one of several JH genes, which are located about 7.7 kilobase pairs from the C mu gene in embryo DNA. The JH-C mu intervening sequence has suffered a deletion of about 2.7 kilobase pairs in HPC-76. Because the delection encompasses sequences required to switch an expressed VH-JH gene from C mu to another CH gene, it may represent a mechanism for "freezing" a lymphocyte clone at the stage of IgM expression. For the second (inactive) C mu allele in HPC-76, the entire joining and switch regions have been deleted; functional inactivation of one allele may thus represent one mechanism by which a lymphocyte clone restricts expression to a single allele (allelic exclusion). Probes generated from the cloned mu gene allowed examination of the JH locus in B, Abelson "pre-B," and T lymphoma cell lines and a myeloid line, all of which cotain RNA species bearing C mu sequences. The B and pre-B lines exhibited recombination within both alleles of the JH locus, suggesting that both alleles may be expressed in some cells. The absence of the JH gene 5' to the recombination sites favors a deletion mechanism for VH-JH joining. Recombination within the JH locus was also detected in two out of four T lymphoma lines, but not in the myeloid line. This indicates that the mechanism by which B cells generate immunoglobulin diversity is operational in some T cells. Lines that synthesize mu RNA without JH rearrangement may have activated the C mu gene directly or have undergone recombination at a more distant locus.

The major intron between the J and C gene segments of the immunoglobulin heavy (H) chain locus contains an enhancer-like sequence, and it has been proposed that this enhancer is necessary to achieve high levels of H chain expression. We have isolated a subclone of the lymphoid pre-B cell line 18-81 that lacks this enhancer but nevertheless produces mu chain at the level characteristic of pre-B cells. Another subclone with a larger deletion does not produce mu chain, but upon fusion with a myeloma that does not produce any immunoglobulin chain, mu chain is expressed by the homolog from the pre-B subclone. The hybridoma lacks the proposed enhancer element in cis; nevertheless it produces as much mu chain as other plasma cell hybridomas. Therefore, this enhancer element is not obligatory for a high level of H chain production.

Complete suppression of expression of immunoglobulin kappa light chain was achieved by injecting female mice from birth with a mixture of antisera against the mu heavy chain and kappa light chain (anti-mu and anti-kappa). Then their offspring were injected with anti-kappa from birth. This resulted in stable suppression as long as anti-kappa injections were continued. kappa light chain was not detectable either in serum or at the cellular level. The number of B cells in spleen and the concentration of immunoglobulin classes and subclasses in serum were normal. The normal levels were achieved by a compensating enhancement of lambda light chain expression. Analysis of the light chains of immunoglobulins secreted by spleen cells from suppressed mice after liposaccharide stimulation by two-dimensional gels showed lambda chain to have a limited heterogeneity. Primary responses to dinitrophenol, influenza strain A, and keyhole limpet hemocyanin were drastically affected, whereas secondary responses appeared to be quite normal, suggesting a surprisingly large potential repertoire.