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The bcl-2 proto-oncogene can prevent the death of many cell types. Mice were generated that were chimeric for the homozygous inactivation of bcl-2. Lymphocytes without Bcl-2 differentiated into phenotypically mature cells. However, in vitro, the mature T cells that lacked Bcl-2 had shorter life-spans and increased sensitivity to glucocorticoids and gamma-irradiation. In contrast, stimulation of CD3 inhibited the death of these cells. T and B cells with no Bcl-2 disappeared from the bone marrow, thymus, and periphery by 4 weeks of age. Thus, Bcl-2 was dispensable for lymphocyte maturation, but was required for a stable immune system after birth.

LPS stimulation of B lymphocytes induces germline transcription of and subsequent switching to the gamma 2b gene. Mature germline transcripts contain an I exon (non-coding) spliced to the C gamma 2b exons. To investigate the role of germline transcription and/or transcripts in heavy chain class switching, we have replaced the germline I gamma 2b promoter and I exon in ES cells with an expressed neomycin resistance gene. The mutated chromosome retains the downstream target sequence for switch recombination (S regions) and all sequences necessary for expression of a switched gamma 2b gene. Wild-type or mutant ES cells were injected into RAG-2 deficient blastocysts to generate somatic chimeras in which all lymphocytes were ES-cell derived. Chimeras derived from injection of heterozygous mutant ES cells had normal levels of serum IgG2b, but their splenic B cells showed a partial decrease in ability to switch to gamma 2b. Strikingly, B lymphocytes from chimeras derived by injection of homozygous mutant ES cells were deficient in IgG2b production both in vivo and in vitro, but normal with respect to production of other Ig heavy chain isotypes. Additional studies demonstrated that lack of ability to produce IgG2b by the mutant B cells correlated with lack of germline transcription and resulted from a specific defect in class-switch recombination to S gamma 2b. Together, these studies demonstrate that the I region is an important regulatory element for control of class-switch recombination.

During the assembly of immunoglobulin and T cell receptor variable region genes from variable (V), diversity (D), and joining (J) segments, the germline-encoded repertoire is further diversified by processes that include the template-independent addition of nucleotides (N regions) at gene segment junctions. Terminal deoxynucleotidyl transferase (TdT)-deficient lymphocytes had no N regions in their variable region genes, which shows that TdT is responsible for N region addition. In addition, certain variable region genes appeared at increased frequency in TdT-deficient thymocytes, which indicates that N region addition also influences repertoire development by alleviating sequence-specific constraints imposed on the joining of particular V, D, and J segments.

Analyses of mechanisms that regulate V(D)J recombination have relied heavily on the use of transformed precursor lymphocyte cell lines. We now show that such lines have highly variable and frequently low levels of recombination activating genes (RAG)-1 and -2 gene expression. We also show that expression levels of the RAG genes can vary > 100-fold between different subcloned cells of an individual pre-B line. We discuss these findings in the context of normal regulation of RAG gene expression and the implication for the use of transformed pre-B cell lines as models for studying control of V(D)J recombination activity.

Background: Mice homozygous for a loss-of-function mutation of the recombination-activating gene-2 (RAG 2), which is required for the rearrangement of antigen receptor genes, do not produce mature B and T lymphocytes. But chimeric mice that result from injection of normal embryonic stem (ES) cells into blastocysts from RAG2-deficient mice develop normal mature lymphocyte populations, all of which are derived from the injected ES cells; we have called this process RAG2-deficient blastocyst complementation. Using ES cells with homozygous mutations, RAG-2-deficient blastocyst complementation could provide a physiological assay with which to determine the potential role of almost any gene in the development and/or function of lymphocytes. To test the general utility of this system, we have used it to test the differentiation-potential of ES cells that harbor homozygous loss-of function mutations of their retinoblastoma susceptibility (Rb) gene loci. We chose Rb for this analysis because of its widespread function in the control of the cell cycle and cell differentiation, the adverse effect of homozygous germline mutations of Rb on hematopoiesis in fetal liver, and the embryonic lethality that results when the homozygous Rb mutation is introduced into the germline., Results: Homozygous Rb mutant ES cells can develop into phenotypically normal, mature B and T lymphocytes in the RAG-2-deficient background. Strikingly, Rb-deficient B and T cells do not have major defects in either activation or function., Conclusion: We have demonstrated the efficacy of the RAG-2-deficient blastocyst complementation system for evaluating the role of critical genes in lymphocyte development. Our results indicate that Rb expression is not intrinsically required for B-cell or T-cell function, despite the normally high levels of Rb expressed in lymphoid cells.

B lymphocyte differentiation is characterized by an ordered series of Ig gene assembly and expression events. In the majority of normal B cells, assembly and expression of Ig heavy (H) chain genes precedes that of light (L) chain genes. To determine the role of the Ig heavy chain protein in B cell development and L chain gene rearrangement, we have generated mice that cannot assemble Ig H chain genes as a result of targeted deletion of the JH gene segments in embryonic stem cells. Mice homozygous for this deletion are devoid of slg+ B cells in the bone marrow and periphery. B cell differentiation in these mice is blocked at the large, CD43+ precursor stage. However, these precursor B cells do assemble kappa L chain genes at a low level in the absence of mu H chain proteins. These data demonstrate that rearrangement and expression of the mu H chain gene is not absolutely required for kappa L chain gene rearrangement in vivo. Expression of mu chains may facilitate either efficient L chain gene rearrangement or the survival of cells that have rearranged light chain genes by promoting the differentiation of large, CD43+ to small, CD43- pre-B cells.

Cells maintain the integrity of their genome through an intricate network of repair systems that recognize and remove lesions from DNA. The only known site-directed recombination process in vertebrates is the V(D)J recombination of lymphocyte antigen receptor genes. A large panel of cell lines deficient in DNA repair were tested for the ability to perform V(D)J recombination after introduction of the RAG-1 and RAG-2 genes. Two mutants failed to generate normal V(D)J recombination, and further analysis provided evidence for two distinct nonlymphoid-specific genes that encode factors involved in both DNA repair and V(D)J recombination.

N- and L-Myc, like c-Myc, contain adjacent basic region (BR), helix-loop-helix (HLH) and leucine zipper (LZ) motifs, which characterize a family of DNA-binding proteins. We have used a polymerase chain reaction (PCR)-based binding site selection technique to demonstrate that the most highly preferred binding site for both N- and L-Myc fusion proteins contains a CACGTG motif, the core binding sequence previously identified for c-Myc. Further analysis identified other N-Myc binding sequences, including asymmetric sequences such as CAT-GTG. N-Myc, like c-Myc, preferentially forms heterodimeric DNA-binding complexes with Max protein. Mutational analyses of N-Myc basic region (BR), helix-loop-helix (HLH) and leucine zipper (LZ) regions revealed that all three regions are necessary for DNA binding by N-Myc-Max complexes, and that dimerization requires both HLH and LZ motifs, while BR sequences are needed only for DNA binding. Our findings support the notion that the LZ motif is a critical element in dimer formation by bHLH-LZ proteins.

The differentiation of B lymphocytes from their progenitors progresses through a series of successive stages that are defined by sequential rearrangement of Ig loci and surface expression of various stage-specific markers, including Ig heavy and light chain proteins. Considerable evidence suggests that the appearance of cells with an orderly progression of Ig gene rearrangements is linked to the expression of the rearranged Ig gene products. Recent experiments have clarified our understanding of mechanisms by which rearrangement of Ig gene segments is controlled and how Ig gene products participate in the regulation of the B-cell differentiation program.

We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals.

Introduction of TCR alpha transgene, TCR beta transgene, or both into RAG-2-/-mice differentially rescues T cell development. RAG-2-/- mice have small numbers of TCR-CD4-CD8-(double negative, DN) thymocytes that express CD3 gamma delta epsilon and zeta proteins intracellularly. Introduction of a TCR beta transgene, but not a TCR alpha transgene, into the RAG-2-/- background restored normal numbers of thymocytes. These cells were CD4+CD8+ (double positive, DP) and expressed small amounts of surface TCR beta chain dimers in association with CD3 gamma delta epsilon but not zeta. RAG-2-/- mice that expressed alpha and beta TCR transgenes developed both DP and single positive thymocytes. Thus, the TCR beta subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition.

Transgenic mice carrying either the c-myc or N-myc oncogene deregulated by the immunoglobulin heavy chain enhancer element (E mu) develop both pre-B and B cell lymphomas (E mu-c-myc and E mu-N-myc lymphomas). We report here that B cell lines derived from these tumors, as well as a line derived from v-myc retroviral transformation, simultaneously express surface immunoglobulin (a hallmark of mature B cells) as well as a common subset of genes normally restricted to the pre-B stage of development-including the recombinase activating genes RAG-1 and RAG-2. Continued RAG-1 and RAG-2 expression in these lines is associated with VDJ recombinase activity detected with a VDJ recombination substrate. Cross-linking of the surface immunoglobulin on these lines with an anti-mu antibody leads to rapid, specific and reversible down-regulation of RAG-1 and RAG-2 gene expression. We also find that a small but significant percentage of normal surface immunoglobulin bearing bone marrow B cells express the RAG-1 gene. These findings are discussed in the context of their possible implications for the control of specific gene expression during the pre-B to B cell transition.

We describe a novel regulatory myosin light chain gene (termed precursor lymphocyte-specific regulatory light chain or PLRLC) that is expressed specifically in precursor B and T lymphocytes. PLRLC is the first example of a regulatory myosin light chain gene which displays specific expression in non-muscle cells. PLRLC is expressed in adult bone marrow derived normal and transformed pre-B cells; in the former, PLRLC expression levels are induced by the pre-B cell specific growth factor interleukin-7 (IL-7). PLRLC is not expressed in either transformed pre-B cells derived from fetal liver or in normal fetal liver pre-B clones grown in the presence of IL-7. Therefore this gene provides the first marker that clearly distinguishes these two pre-B subsets. Finally, several of the different PLRLC transcripts potentially encode regulatory myosin light chains with unique structural features. The unique distribution, regulation and structural features of the PLRLC gene products suggest an important role for PLRLC during lymphocyte development.

Mouse plasmacytomas induced by pristane oil alone, or in combination with Abelson murine leukemia virus (A-MuLV), regularly carry one of three alternative chromosomal translocations that juxtapose c-myc to immunoglobulin heavy- or light-chain loci. E mu-c-myc transgenic mice develop translocation-free plasmacytomas after induction by pristane oil and/or A-MuLV [Sugiyama, H., Silva, S., Wang, Y., Weber, G., Babonits, M., Rosen, A., Wiener, F. & Klein, G. (1990). Int. J. Cancer, 46, 845-852]. In order to test whether another member of the myc family, N-myc, could play a similar role as c-myc, we treated E mu-N-myc transgenic mice with pristane and helper-free A-MuLV. Of 20 mice that received a single pristane injection followed by A-MuLV, 17 developed plasmacytomas with a mean latency period of 54 +/- 20 days. In a corresponding group that only received a single pristane injection, five out of six transgenic mice developed plasmacytomas with a mean latency period of 142 +/- 32 days. However, after three monthly injections of pristane, all 15 transgenic mice developed plasmacytomas with a mean latency period of 128 +/- 20 days. All plasmacytomas expressed the N-myc transgene, while none of them expressed either c-myc or endogenous N-myc. None of the tumors carried the usual plasmacytoma-associated translocations.

The RAG-1 and RAG-2 genes synergistically confer VDJ recombinase activity to nonlymphoid cell lines. To unequivocally test RAG gene function, we created lines of mice that lack functional copies of these genes. Consistent with the possibility that RAG gene encode the tissue-specific components of VDJ recombinase, RAG-2-deficient mice are viable but have a severe combined immune deficiency due to inability to initiate VDJ recombination and thereby generate mature lymphocytes. RAG-2-deficient mice have no obvious defect in any tissue or lineage other than lymphocytes, indicating that VDJ recombinase activity and RAG-2-gene function is required only for lymphocyte development. Levels of RAG-1 and RAG-2 expression in primary murine lymphoid tissues and lymphoid bone marrow cultures generally are much higher than those of transformed precursor B-cell lines. Low-level RAG gene expression in permanent cell lines results from a decline during propagation due to outgrowth of cells with lower RAG expression levels. The low and variable level of RAG gene expression in transformed pre-B cell lines correlates with low and variable rates of endogenous VDJ recombination; therefore, such lines are not reliable models for experiments aimed at studying mechanisms that target this activity to particular variable region gene segments. To generate such a system, we introduced RAG genes into B-lineage lines under the control of a heat shock-inducible promoter; heat-shock treatment induces extremely high-level but transient RAG expression accompanied by parallel induction of VDJ recombinase activity. Such cells efficiently rearrange transfected VDJ recombination substrates in a regulated manner that is dependent on the activity of transcriptional control elements associated with the target V gene segments.

The site-specific recombination mechanism responsible for the assembly of antigen receptor variable region genes is only employed in lymphocyte development. Gene-targeted and other types of mutational analyses have implicated at least seven distinct gene products, some lymphoid-specific and others more generally expressed, as involved in aspects of this process. Mutation of the lymphocyte-specific activities required for VDJ recombination or mutation of the target gene segments of this process have created B and/or T cell-deficient mouse models that have provided new insights into the regulation of the VDJ recombination reaction and into how the successful achievement of this reaction at various loci helps lead lymphocytes through their early developmental program A second type of B lymphocyte-specific recombination process is involved in heavy-chain class switching; gene-targeted mutation approaches also have provided new insights into the cis-acting elements that target of this reaction to particular CH genes.

X-linked agammaglobulinemia (XLA) patients manifest a very low production of immunoglobulins (Ig) of all classes and plasma cells are virtually absent. The XLA gene plays a crucial role in the transition of pre-B cells to later B cell stages, as hardly any slg-positive B lymphocytes can be detected. In the bone marrow almost normal numbers of pre-B lymphocytes are present. These cytoplasmatic C mu+ pre-B lymphocytes appear to express truncated M heavy chain molecules lacking the variable region segment. The T lymphocyte compartment is intact: the numbers of mature T cell receptor (TcR) alpha beta expressing T lymphocyte populations and their proliferative responses to antigens are normal. That the B cells are primary and exclusively affected was proven by X-chromosome inactivation studies. There is no evidence that the XLA gene is directly involved in the Ig gene rearrangements since B lymphoblastoid cell lines (BLCLs) established from peripheral blood of XLA patients were found to produce IgM molecules composed of complete Ig heavy and light chains and were shown to contain normal VHDJH recombinations. The data do not exclude the involvement of the XLA gene in a B cell specific process that makes the Ig loci accessible for recombination. Investigations on the degree of diversity of immunoglobulins generated by XLA patients exposed no limitations in the VH family usage. Sequence analysis of expressed VH3 and VH4 rearrangements however revealed that some genetic elements of the Ig locus might be over-represented and that a high portion of rearrangements was generated by unconventional mechanisms. By restriction length polymorphism (RFLP) and pulsed field gel electrophoreses analyses the XLA gene was mapped to an 8- to 12-Mb DNA fragment located in the Xq22 region. The known location of the XLA gene on the X-chromosome with closely linked RFLP markers and the availability of X-chromosome inactivation assays provides methods for carrier detection and prenatal diagnosis.

Transgenic mice that contain the L-myc gene under the control of the immunoglobulin heavy-chain enhancer (E mu) express the transgene preferentially in T cells, develop thymic hyperplasia and are predisposed to T-cell lymphomas. An analogous E mu N-myc transgene is expressed preferentially in pre-B and B cells and provokes the development of B-cell neoplasias. Animals with an E mu pim-1 construct express the transgene in both B and T cells, but succumb to T-cell lymphomas. Complementation of the E mu N- and L-myc transgenic mice by breeding with E mu pim-1 animals leads to much more rapid development and a dramatically higher incidence of lymphoid malignancies, but the lineage specificity prescribed by the E mu N- and L-myc transgenes is maintained. The different oncogenic potential of myc genes is illustrated by the average latency period of tumor manifestation in double transgenics. Whereas c-myc/pim-1 animals develop pre-B-cell leukemia prenatally, the mean latency period for N-myc/pim-1 and L-myc/pim-1 mice is 36 and 94 days respectively. The N- and L-myc transgenes are expressed at high levels in tumors from double transgenic mice, but expression of the endogenous c- and N-myc genes is undetectable, directly implicating the myc transgenes in the tumor formation process.

X-linked agammaglobulinemia (XLA) is characterized by an arrest in early B lymphocyte differentiation. Precursor B cells are present in the bone marrow (BM), whereas peripheral blood B cell numbers are severely decreased. A series of Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (BLCL) was established from peripheral blood of three XLA patients belonging to one pedigree. These BLCL manifested productive VHDJH rearrangements and a random utilization of the VH families. The CDR3 regions of the rearrangements varied in length from 12 to 47 nucleotides and included N regions in all cases. The results supported the conclusion that the few B lymphocytes in peripheral blood of XLA patients exhibit all mechanisms that generate immunoglobulin (Ig) heavy (H) chain diversity. However, no evidence for somatic mutation was found. Within the VH3 family 50% of the expressed VH gene segments belonged to a single subgroup and within the VH4 family a preferential utilization of one VH4 gene element was observed. The utilization of H chain joining (HH) elements was biased to JH4 and JH6 and a high percentage of the CDR3 regions was found to be generated by unconventional mechanisms, such as multiple D usage and the fusion of D elements to D segments with irregular recombination recognition signals. These unique features of the recombined and expressed VHDJH regions in XLA may explain the inability of XLA patients to respond to a variety of antigens. Alternatively, they could be secondary to a B lymphocyte maturation defect in XLA.

The immunoglobulin light chain V kappa 1 gene family is polymorphic in murine inbred strains and this family has been subdivided into five sub-groups (V kappa 1A-E). The V kappa 1A sub-group contributes to approximately 2% of the total serum immunoglobulin light chains in several mouse strains. However, it has been reported that this sub-group is absent in New Zealand Black (NZB) mouse serum. Amino acid sequencing of myeloma proteins from this inbred mouse has shown that they belong to the V kappa 1B sub-group. We report here the structure of nine functional germline genes from NZB mice that have high homologies to the V kappa 1A, V kappa 1B, V kappa 1C, and V kappa 1D sub-groups. In addition, a novel germline gene representing the prototype of a new sub-group (designated V kappa 1F) has been identified. We have isolated different V kappa 1 germline genes from a single restriction fragment length polymorphism (RFLP) fragment, as well as identical V genes from two different RFLP migrating bands. Therefore, the complexity of the genes encoding the immunoglobulin variable region cannot be determined solely by RFLP analysis. Nucleotide sequence analysis of 16 V kappa 1 genes which code for NZB autoantibodies indicate that they belong to five different V kappa 1 sub-groups with five hybridomas (31%) expressing the V kappa 1A sub-group. Comparison of the sequences of V kappa 1 genes expressed in hybridomas with corresponding germline genes show no somatic mutations.

Preferential usage of JH-proximal VH genes has been demonstrated in immature murine B cell repertoires. To determine whether this phenomenon is also evident in human repertoires, we studied utilization of VH6, the most JH-proximal human VH gene. Examination of VH gene usage in a panel of precursor B cell acute lymphoblastic leukemia samples indicated that 15% of the IgH rearrangements utilized VH6. VH6 is a single-member family in a total repertoire of 100-200 VH genes; thus, if usage were purely random, one would expect VH6 rearrangement frequency to be less than 1%. Analysis of VH gene usage in normal lymphoid tissues also revealed biased usage of VH6. VH6 was preferentially utilized in 16- to 24-week-old fetal liver as compared to adult peripheral blood mononuclear cells or spleen. Possible implications of the conservation of preferential usage of JH-proximal genes in both immature murine and human repertoires are discussed.

The NZB mouse is genetically predisposed to the development of autoimmune disease that resembles the human autoimmune systemic lupus erythematosus and autoimmune hemolytic anemia, with increased titers of anti-DNA, and Coombs' autoantibodies. The various autoimmune traits are controlled separately by a limited number of genes. Genetic studies have shown that several immune loci are involved in autoimmunity: T cell abnormalities, H-2 complex and immunoglobulin genes have been implicated. In this report, we present evidence for a significant correlation of NZB V kappa 1 haplotype defined by restriction fragment length polymorphism analysis with anti-erythrocyte autoantibodies in NZB x 129/J and NZB x SM/J recombinant inbred lines.

The mercury concentrations in the hair of 53 dentists, 49 dental assistants, 35 dental students and 35 non-exposed persons were analyzed by AAS and compared with a group of 22 factory workers producing chloride gas. The average mercury concentrations in the hair of the dentists was found to be twice as high as the amount measured in the non-exposed population, but only one quarter of that established for the factory workers. The dental assistants had 50% more mercury in their hair than the non-exposed group. The mercury concentrations did not correlate with age, sex, or number of amalgam fillings, but rather with the daily/weekly consumption of fish. The mercury concentrations in the hair of dentists and dental personnel found in this study were low, even when compared with international results; toxicologically their importance is negligible.

We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionally active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial beta-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects beta-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector. We have also used the lacZ vector to isolate variant pre-B-cell lines with low and high levels of VDJ recombinase activity.

Expression of the myc family of cellular proto-oncogenes is critical for determining the proliferative, differentiative, and oncogenic potential of a wide variety of cell types. Despite a large body of genetic and biochemical data indicating that myc proteins are located in the nucleus and can bind to nucleic acids, the mechanism by which these proteins exert their effects remains a mystery. The recent observation that myc proteins contain two structural domains previously identified in transcription factors and differentiation factors, the leucine zipper domain and the helix-loop-helix motif, supports the notion that these proteins directly regulate gene expression. In this review we consider the possible significance of these domains to myc function.

Elucidation of the cellular and molecular mechanisms which determine the expressed antibody repertoire remains a major challenge in immunology. Knowledge of V gene diversity, organization, and expression is important to an understanding of the formation of the antibody repertoire in normal as well as diseased states. In the last few years, great advances have been made in our understanding of the human heavy chain variable region (VH) gene locus. In this review we present the current knowledge of VH gene diversity, organization, and utilization in normal individuals followed by a discussion of the possible relevance of these findings to autoimmunity.

We have characterized the structure and expression of transcripts synthesized from the murine germline immunoglobulin gamma 3 heavy chain gene in certain B-lineage cells. The transcripts initiate upstream of the switch gamma 3 region, generating a 5' exon that is spliced to C gamma 3 exons. Expression of this germline transcript is induced when splenic B cells or A-MuLV-transformed pre-B cell lines are cultured in the presence of lipopolysaccharide (LPS). Addition of interleukin-4 (IL-4) to these lipopolysaccharide (LPS) cultures dramatically inhibits induction of the germline gamma 3 transcript. Induction of germline gamma 3 transcripts occurs before the increased accumulation of gamma 3-producing cells and VDJ-gamma 3 mRNA in cultures of splenic B cells. These data provide further evidence that germline CH transcriptional units are important components in the regulation of heavy chain class-switching. In addition, the pre-B cell lines that we describe represent the first example of permanent cell lines that regulate expression of the germline gamma locus in response to LPS plus IL-4 treatment in a manner analogous to normal B cells; therefore these lines should represent an excellent model system to further study the molecular mechanisms by which germline expression is regulated by these agents.

The myc family of nuclear oncogenes contains three well-characterized members, c-myc, N-myc, and L-myc. These genes encode related but distinct nuclear proteins that can contribute to tumorigenic conversion both in vitro and in vivo. However, each gene displays a unique activation pattern in spontaneously arising tumors, a pattern that partially reflects the unique expression pattern of each gene during normal development. Although the specific function of myc family genes has not been determined, changes in myc gene expression accompany in vitro exposure to growth and differentiation stimuli, suggesting an important role in these cellular processes. In addition, the homologies shared among myc genes and two groups of DNA binding, transcriptional regulatory proteins suggests a role in regulating gene expression. This review describes the similar and unique properties of individual members of the myc gene family with respect to their expression and potential role during normal development and in the tumorigenic conversion process.

To elucidate the mechanism of VH to VHDJH joining (VH replacement), we have assayed a pre-B cell line for ability to perform V(D)J recombinase-mediated rearrangement events between an unrearranged VH gene segment and a VHDJH rearrangement within a retroviral recombination substrate. V(D)J recombinase-mediated inversional joins between the VH gene segment and the VHDJH rearrangement within this substrate activate a drug-resistance gene allowing selection of cells that performed the event. We have used this method to identify and isolate VH to VHDJH joins within the introduced substrate. Analyses of the resultant signal and coding joins of such rearrangements demonstrated that VH replacement recombination is mediated by a heptamer-like sequence embedded in the 3' region of the VH segment in the assembled VHDJH join and the heptamer-spacer-nonamer recognition signal of the unrearranged VH segment.

We describe transgenic mice that carry an antigen receptor gene minilocus comprised of germline T cell receptor (TCR) beta variable gene elements (V, D and J) linked to an immunoglobulin (Ig) C mu constant region gene with or without a DNA segment containing the Ig heavy chain transcriptional enhancer (E mu). Transgenic constructs lacking the E mu-containing segment did not undergo detectable rearrangement in any tissue of six independent transgenic lines. In contrast, transgenic constructs containing this DNA segment underwent rearrangement at high frequency in lymphoid tissues, but not other tissues, of four independent lines. Analyses of purified B and T cells, as well as B and T cell lines, from transgenic animals demonstrated that the E mu-containing segment within the construct allowed partial TCR gene assembly (D to J) in both B and T cells. However, complete TCR gene rearrangement within the construct (V to DJ) occurred only in T cells. Therefore, we have demonstrated elements that can control two separate aspects of TCR beta VDJ rearrangement within this construct. One lies within the E mu-containing DNA segment and represents a dominant, cis-acting element that initiates lymphoid cell-specific D beta to J beta rearrangement; various considerations suggest this activity may be related to that of the E mu element. The second element provides T cell-specific control of complete (V beta to DJ beta) variable region gene assembly; it correlates in activity with expression of the unrearranged V beta segment.

A cell line that switches from mu to gamma 2b synthesis during growth in culture uses the same VH region for both heavy chains but retains two copies of the Cmu gene. This suggests that the mu to gamma 2b class switch can occur, at least in part, by an RNA processing mechanism. Regulatory variants of this cell line lose constitutive mu-chain synthesis but simultaneously acquire lipopolysaccharide (LPS)-inducible synthesis of that chain. This co-variation is allele-specific and is correlated to a large deletion of DNA in the JH--Cmu intron.

Seven DNA fragments which map to or very near human chromosome band 2p24 are shown to be differentially amplified in DNA from specific subsets of an enlarged series of human neuroblastoma cell lines and primary neuroblastomas. Of these DNA fragments, the probe NB-19-21 for the oncogene N-myc is the most frequently amplified, with a second expressed sequence (pG21) amplified in 9 of those 11 cell lines and 16 of those 25 tumors exhibiting amplification of N-myc. The remaining probes are in turn each amplified in progressively smaller, nested subsets of the cell lines and tumors in which both N-myc and pG21 are amplified. These data permit construction of models for the organization of a "neuroblastoma amplicon," i.e., an originally amplified DNA domain, with N-myc positioned most central and the other DNA fragments increasingly peripheral; comparable models result for the cell lines and the tumors. Five of the seven probes examined detect novel DNA fragments in these specimens, reinforcing previous observations that extensive DNA rearrangement can occur during DNA amplification in neuroblastoma cell lines and in primary neuroblastomas. Such rearrangements could contribute significantly to the evolution of the neuroblastoma amplicon in different specimens to progressively smaller units, preserving, in the limit, amplification of N-myc.

We have studied frequencies of VH gene utilization in a panel of monoclonal Epstein-Barr virus (EBV)-transformed B cell lines derived from human adult and fetal tissues as well as in monoclonal B cells obtained from fresh chronic lymphocytic leukemia (CLL) samples. The results show that IgM-secreting EBV cell lines from both fetal and adult tissues utilize VH genes from particular families roughly in proportion to estimated family size, suggesting that the repertoire of sigM-positive B cells in both fetal and adult organs is 'normalized' with respect to the V(H) gene family. In contrast, we find a highly biased pattern of VH gene expression in CLLs. The significance of these findings is discussed in the context of mechanisms that could be involved in normal B cell repertoire development and in the process of malignant transformation of precursors of CLL.

Amplification of clones 8, G21, and N-myc, which were derived from human neuroblastoma cell lines IMR-32 and NB-19, were studied in nine neuroblastoma xenografts. N-myc was amplified from 50- to 120-fold in eight of nine xenografts, clone 8 was amplified in five of the xenografts, and clone G21 was amplified in four of these five. Each of these clones was localized by in situ hybridization to homogeneously staining regions in metaphase spreads of xenograft chromosomes. In one xenograft a DNA rearrangement of clone 8 was observed, and only two of the sequences detected by G21 were amplified. Restriction enzyme mapping indicated that the rearrangement within clone 8 occurred at a position close to the rearrangement previously noted in neuroblastoma cell line NB-9.

In neuroblastoma lines in which the N-myc gene is present as a single copy, the expression of N-myc as messenger RNA is increased relative to that in nonneuroblastoma cell lines and tumors. The increase of expression in neuroblastomas with amplified N-myc genes is the result of (i) an increase in the absolute amount of expression of each N-myc gene and (ii) an increase in the copy number of the N-myc gene. A second gene--which is amplified in many of the same lines as N-myc--is expressed to about the same degree in most human cell lines and primary tumors regardless of origin (when normalized to gene copy number). Thus, a change in the regulation of N-myc expression in neuroblastomas and certain other tumors results in greatly increased expression of each N-myc gene copy.

We have cloned a 2.0-kb EcoRI fragment of human genomic DNA (NB-19-21) which has homology to the v-myc oncogene but is distinct from the classical c-myc gene. This sequence is amplified from 25- to 700-fold in eight of nine tested human neuroblastoma cell lines which contain either homogeneously staining regions or double minutes (HSRs or DMs), the caryological manifestations of amplified genes. In the remaining line, the c-myc proto-oncogene is amplified approximately 30-fold. NB-19-21 hybridizes to a 3.2-kb cytoplasmic, poly(A)+ RNA species that is abundant only in lines in which the sequence is amplified. We propose that the gene encoding the NB-19-21-related RNA species may represent a new oncogene, which we call N-myc. NB-19-21 derives from chromosome 2; but in the five HSR-containing lines that have amplified this sequence, none has HSRs on chromosome 2. NB-19-21 is associated with DMs in a DM-containing line. A second, randomly cloned, amplified DNA segment from the HSR of one of the neuroblastoma lines is amplified in a subset of the lines in which NB-19-21 is amplified. In addition, this probe identifies a novel joint in the amplification unit of one line relative to that of the others. We suggest that, in the eight lines which have amplified NB-19-21, the amplification units are overlapping, but not identical, and that transposition of the common sequences may occur prior to amplification.

A cell line transformed by Abelson murine leukemia virus, called PD, is capable of carrying out kappa-gene rearrangement while growing in culture. Subclones of PD have diverse kappa-gene structures, and some derivatives show evidence of continued joining activity after as many as three subclonings. Analysis of PD sublineages has shown that a rearranged chromosome can undergo secondary kappa-gene rearrangements, producing either a new rearrangement or a deletion of C kappa. Although the PD line actively rearranges its kappa genes, its rearranged heavy-chain genes show little variation, and there is no rearrangement of lambda genes. In PD subclones, DNA fragments representing the reciprocal product of kappa-gene rearrangement are often evident, and they may undergo either further rearrangement or deletion. The implications of multiple rearrangements on a single chromosome and of the maintenance of reciprocal fragments are considered in the context of a model that postulates that the V kappa and J kappa segments are not all organized in the DNA in the same transcriptional direction, leading to inversions rather than deletions during joining.

We investigated the immunochemical and molecular characteristics of murine monoclonal rheumatoid factors. Study of the fine specificity of 20 monoclonal rheumatoid factor antibodies shows a wide degree of heterogeneity. However, many express an interstrain cross-reactive idiotype. We show that our rheumatoid factors utilize a restricted set of the heavy-chain variable region (VH) repertoire representing the more 3' VH families. Preferential expression of 3' VH families is known to occur early in development. We report the nucleotide sequence of two cloned rheumatoid factor VH genes, Y19-10 (VH J558) and 129-48 (VH 7183) in which no major differences are observed between VH genes encoding the heavy chain of autoantibodies and antibodies against foreign antigens.

The immunoglobulin heavy chain variable region is encoded as three separate libraries of elements in germ-line DNA: VH, D and JH. To examine the order and regulation of their joining, we have developed assays that distinguish their various combinations and have used the assays to study tumor cell analogs of B-lymphoid cells as well as normal B-lymphoid cells. Abelson murine leukemia virus (A-MuLV) transformed fetal liver cells - the most primitive B-lymphoid cell analog available for analysis - generally had DJH rearrangements at both JH loci. These lines continued DNA rearrangement in culture, in most cases by joining a VH gene segment to an existing DJH complex with the concomitant deletion of intervening DNA sequences. None of these lines or their progeny showed evidence of VHD or DD rearrangements. Heavy chain-producing tumor lines, representing more mature stages of the B-cell pathway, and normal B-lymphocytes had either two VHDJH rearrangements or a VHDJH plus a DJH rearrangement at their two heavy chain loci; they also showed no evidence of VHD or DD rearrangements. These results support an ordered mechanism of variable gene assembly during B-cell differentiation in which D-to-JH rearrangements generally occur first and on both chromosomes followed by VH-to-DJH rearrangements, with both types of joining processes occurring by intrachromosomal deletion. The high percentage of JH alleles remaining in the DJH configuration in heavy chain-producing lines and, especially, in normal B-lymphocytes supports a regulated mechanism of heavy chain allelic exclusion in which a VHDJH rearrangement, if productive, prevents an additional VH-to-DJH rearrangement.

We demonstrated that a subclone of an Abelson murine leukemia virus-transformed B-lymphoid cell line switched from mu to gamma 2b expression in vitro, by the classical recombination-deletion mechanism. In this line, the expressed VHDJH region and the C gamma 2b constant region gene were juxtaposed by a recombination event which linked the highly repetitive portions of the S mu and S gama 2b regions and resulted in the loss of the C mu gene from the intervening region. An additional recombination event in this subclone involved an internal deletion in the S mu region of the expressed (switched) allele. One end of this deletion occurred very close to the switch recombination point. Despite the recombination-deletion mechanism of switching, the gamma 2b-producing line retained two copies of the C mu gene and two copies of the sequence just 5' to the S gamma 2b recombination point. The possible significance of the retention of these sequences to the mechanism of class switching is discussed.

The most JH-proximal VH gene segments are used highly preferentially to form VHDJH rearrangements in pre-B-cell lines. This result demonstrates that the rate at which immunoglobulin VH gene segments recombine is influenced by their chromosomal organization, and that the initial repertoire of VH genes expressed in pre-B cells is strikingly different from that seen in mature populations.

To elucidate mechanisms that regulate ordered and tissue-specific assembly of Ig and TCR variable region gene segments, we have introduced a recombination substrate comprised of germline TCR beta V, D, and J gene segments into an Abelson murine leukemia virus-transformed pre-B cell line that actively rearranges endogenous Ig H chain variable region gene segments but does not rearrange endogenous light chain or TCR variable region gene segments. We find that these cells efficiently join D beta segments to J beta segments within the mini-locus, but that they do not make any detectable site-specific rearrangements of the introduced V beta segment even though it is closely linked in the same construct to the D beta. These findings suggest that factors necessary for V beta to (D beta)J beta joining may be absent in these pre-B cells and also imply that the order in which TCR V beta, D beta, and J beta segments are rearranged can be influenced by factors other than the 12/23 recombination rule. Furthermore, in agreement with the an accessibility model of VDJ recombinase control, the D beta region of the construct was found to be relatively more sensitive to DNAase I digestion in isolated nuclei when compared to the unrearranged V beta region.