Immunoglobulin Kappa Light Chain Genes Research Articles

Immunophenotypic and gene probe analysis of a case of Richter's syndrome.

A rare case of diffuse large cell lymphoma (DLL) arising in the stomach of a patient with long-standing chronic lymphocytic leukemia (CLL) has been examined by a combination of immunophenotypic and gene probe methods. Both the resected lymphoma and the CLL (obtained from an infiltrate in perigastric lymph nodes) expressed surface membrane markers consistent with a B-cell origin (HLA-DR+; B1+; B4+), but surface membrane immunoglobulin (SmIg) could be detected only on the CLL cells (SmIgM lambda+). Neither neoplasm expressed cytoplasmic immunoglobulin. DNA probe analysis using the Southern blot method and probes to the joining (J-H) region of the immunoglobulin heavy chain gene and to the constant (C) regions of the immunoglobulin kappa and lambda light chain genes revealed different light and heavy chain gene rearrangements in the two neoplasms. These findings suggest that the large cell lymphoma arose from a different clone than the CLL and the development of large cell lymphoma may have represented a de novo neoplastic event in a predisposed person.

Determinant differences between the rabbit and mouse immunoglobulinxenhancers impair the activity of the rabbit enhancer in mouse myeloma cells

Enhancer activity of the rabbit immunoglobulin kappa light chain gene intron conserved region (KICR) was examined in mouse myeloma cells using transient expression experiments. Compared to the homologous region of the mouse kappa light chain gene, the rabbit KICR shows nearly no stimulatory effect on expression of the indicator gene, cat. Experiments with mouse-rabbit chimeric KICRs indicated that differences in the region around the NF-kappa B binding site are responsible for the impaired activity of the rabbit KICR whereas mouse sequences covering the kappa E2 and kappa E3 motifs can be replaced by the equivalent rabbit fragment without affecting enhancer function. Creation of a perfect mouse NF-kappa B target sequence in the rabbit gene only partially restores enhancer activity. Furthermore, mouse and rabbit DNA fragments encompassing the NF-kappa B target sequence behave in an identical manner in an electrophoretic mobility shift assay. The results indicate species-related functional differences in the immunoglobulin kappa light chain gene enhancer and suggest that although the NF-kappa B binding site plays a crucial role in enhancer activity surrounding gene elements are also necessary for full enhancer effect.

NF-kappa B protein purification from bovine spleen: nucleotide stimulation and binding site specificity.

The activity of the enhancer for the kappa immunoglobulin light chain gene critically depends on the presence in the nucleus of the NF-kappa B protein. We purified NF-kappa B over 50,000-fold and identified two protein species, 42 and 44 kDa, that could be eluted and renatured from a sodium dodecyl sulfate/polyacrylamide gel to give specific DNA-binding activity. Binding of the purified bovine NF-kappa B as well as that from human and murine B- or T-lymphoid cell extracts was dramatically stimulated by nucleoside triphosphates. This effect distinguished NF-kappa B from a related factor, H2-TF1. Purified NF-kappa B interacted efficiently with regulatory sequences that function during either B- or T-lymphocyte activation, including the human immunodeficiency virus enhancer and a NF-kappa B binding site we detected in the interleukin 2 enhancer.

I kappa B: a specific inhibitor of the NF-kappa B transcription factor.

In cells that do not express immunoglobulin kappa light chain genes, the kappa enhancer binding protein NF-kappa B is found in cytosolic fractions and exhibits DNA binding activity only in the presence of a dissociating agent such as sodium deoxycholate. The dependence on deoxycholate is shown to result from association of NF-kappa B with a 60- to 70-kilodalton inhibitory protein (I kappa B). The fractionated inhibitor can inactivate NF-kappa B from various sources--including the nuclei of phorbol ester-treated cells--in a specific, saturable, and reversible manner. The cytoplasmic localization of the complex of NF-kappa B and I kappa B was supported by enucleation experiments. An active phorbol ester must therefore, presumably by activation of protein kinase C, cause dissociation of a cytoplasmic complex of NF-kappa B and I kappa B by modifying I kappa B. this releases active NF-kappa B which can translocate into the nucleus to activate target enhancers. The data show the existence of a phorbol ester-responsive regulatory protein that acts by controlling the DNA binding activity and subcellular localization of a transcription factor.

Modern evolution of a single-copy gene: the immunoglobulin C kappa locus in wild mice.

The immunoglobulin kappa light-chain constant region gene (C kappa) has been cloned and sequenced from five wild mouse species. Analysis of these data has permitted an assessment of single-copy gene evolution during a limited time period as defined by the genus Mus. Sequence conservation was found to be as high (or higher) in the 5' and enhancer regions as in the coding region. The pattern of substitutions throughout these genes suggests that parallel evolution has occurred frequently and that substitutions at replacement sites have not decreased significantly, owing to saturation during this period of approximately 10 Myr. Phylogenetic relationships have been determined among these wild species as well as among members of the genus Rattus.

Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immunoglobulin heavy chain enhancer.

Transgenic rabbits with the rabbit c-myc oncogene fused with the rabbit immunoglobulin heavy chain enhancer region (E mu) DNA were developed by microinjecting pronuclei of single cell zygotes with the gene construct and implanting the microinjected eggs into pseudopregnant females. At age 17-20 days, 3 of 21 offspring born to seven females were found to have peripheral blood leukocyte counts of greater than 100,000 per mm3. Histology analyses showed extensive lymphocytic infiltration in the liver and kidney, indicating that these rabbits had a malignant lymphocytic leukemia. Genomic DNA analyses of thymus and peripheral blood lymphocytes revealed that the leukemic rabbits were transgenic and that both immunoglobulin heavy and kappa light chain genes were rearranged in the leukemic cells; thus, the leukemic cells are of B-cell lineage. One to four heavy and light chain gene rearrangements were observed, suggesting that the B-cell tumors were oligoclonal. Stable tissue culture cell lines from the bone marrow and peripheral blood of one of the transgenic rabbits have been developed. The development of B-cell leukemias in rabbits with the E mu-myc transgene contrasts with the development of B-cell lymphomas in mice carrying a similar transgene. The lymphomas in mice develop in adults and are monoclonal in origin. The leukemias in rabbits develop in juveniles, less than 3 weeks after birth, and appear oligoclonal in origin. The leukemias seem to develop in rabbit at a specific stage of development, and we suggest that a normal developmental signal may be involved in the oncogenesis.

Oligonucleotide that binds nuclear factor NF-kappa B acts as a lymphoid-specific and inducible enhancer element.

The immunoglobulin kappa light chain gene contains a lymphoid-specific enhancer that includes several short protein-binding sequences. The sequence that binds the nuclear factor NF-kappa B was tested for its ability to act independently as an enhancer element by inserting it into test plasmids containing the chloramphenicol acetyltransferase gene. When analyzed for activity by transient transfection into lymphoid and nonlymphoid cells, a single copy of the NF-kappa B binding site could act as a tissue-specific upstream activating element. Two copies (dimer) showed 10-fold higher activity than did one copy and could act as an enhancer element 2.5 kilobases downstream of the transcriptional start site. The enhancer activity of this sequence was correlated with the presence of the cognate binding protein, NF-kappa B. This sequence acted as an inducible enhancer under conditions that induce NF-kappa B binding activity. Thus, the NF-kappa B binding site acts by itself as a tissue-specific and inducible enhancer element, and two copies show cooperative interaction.

Two transcription factors, NF-kappa B and H2TF1, interact with a single regulatory sequence in the class I major histocompatibility complex promoter.

A sequence centered 166 nucleotides upstream of the mouse H-2Kb class I major histocompatibility gene binds a nuclear factor, H2TF1, found in many cell types. Previous studies have shown that binding of H2TF1 to this sequence stimulates class I gene expression. Furthermore, this factor binds a similar sequence in the 72-base-pair repeat enhancer element of simian virus 40. We show here that NF-kappa B, an inducible B-cell-specific factor that binds the kappa immunoglobulin light chain gene enhancer, also binds the H2TF1 regulatory sequence. Methylation-interference experiments demonstrate that NF-kappa B closely interacts with six of the eight symmetrically positioned guanines that contact H2TF1. These experiments suggest that NF-kappa B may play a role in class I major histocompatibility gene expression and that H2TF1 and NF-kappa B may be related DNA-binding proteins.

The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated.

Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.

The Coupling Between Enhancer Activity and Hypomethylation of κ Immunoglobulin Genes is Developmentally Regulated

Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.

Expression in transgenic mice of two genes of different tissue specificity integrated into a single chromosomal site.

Transgenic mice were used to study the expression of pairs of genes with distinctly different tissue specificities, covalently linked and integrated into the same chromosomal site. A transgenic strain carrying, in close proximity and in the same orientation, the rat fast skeletal muscle myosin light-chain 2 (MLC2) gene and the mouse rearranged immunoglobulin kappa light-chain gene expressed the immunoglobulin gene specifically in the lymphoid tissues, whereas rat MLC2 transcripts were found in skeletal muscle but not in the spleen or the other tissues that were tested. In another transgenic strain, carrying the rat MLC2 gene and a modified rat skeletal muscle actin gene (actin-globin chimeric gene), transcripts of the rat MLC2 gene were detected in skeletal muscle only, whereas the actin-globin transcripts were detected in skeletal muscle as well as in the heart. Moreover, the expression of the chimeric gene was also developmentally regulated. Expression was higher in cardiac muscle than in the skeletal muscle of neonatal mice, whereas expression was higher in skeletal muscle in adult mice. This pattern is consistent with the regulation of the expression of the endogenous skeletal muscle actin gene. Thus, in those transgenic strains that expressed both genes, each gene retained its tissue specificity, in spite of their close proximity. These results indicate a high degree of autonomy of the control elements included in the cloned genomic DNA fragment and demonstrate that a single chromosomal site can be permissive for the proper expression of two genes with different tissue specificities.

Lymphoma cell line (FL-18) and Epstein-Barr virus-carrying cell line (FL-18-EB) obtained from a patient with follicular lymphoma: monoclonal derivation and different properties

A novel cell line, FL-18, was established from the pleural effusion of a patient with follicular small cleaved cell lymphoma. At the same time, an Epstein-Barr virus (EBV) nuclear antigen (EBNA)-positive cell line, FL-18-EB, was established from the EBV-infected culture of the same pleural effusion cells. Both cell lines had the same monoclonal surface immunoglobulin (IgG kappa), and they had the same karyotype as that of the fresh pleural effusion cells in which a reciprocal translocation between the long arm of chromosomes 14 and 18 [t(14;18)(q32;q21)] was detected. Gene rearrangement analysis of immunoglobulin heavy-chain gene (JH) and kappa light-chain gene (J kappa) showed the same rearranged configuration in the two cell lines; however, some morphological and phenotypic differences were found. The FL-18-EB cells, which were morphologically similar to common EBNA- positive lymphoblastoid cell lines of normal B cell origin at the initial phase of culture, were larger than the FL-18 cells and contained multinucleated giant cells. The FL-18 cells lacked cytoplasmic immunoglobulin and were positive for common acute lymphoblastic leukemia antigen (CALLA), whereas the FL-18-EB cells had cytoplasmic immunoglobulin and were negative for CALLA. Thus, the phenotype of FL-18-EB seems to be a result of a shift by EBV infection to a more mature stage in the B cell differentiation pathway than that of FL-18. The paired availability of EBV-free and EBV-infected cell lines of a neoplastic clone is unique and valuable in considering EBV infectibility of neoplastic B cells and resultant phenotypic changes.

Lymphoma Cell Line (FL-18) and Epstein-Barr Virus–Carrying Cell Line (FL-18-EB) Obtained From a Patient With Follicular Lymphoma: Monoclonal Derivation and Different Properties

A novel cell line, FL-18, was established from the pleural effusion of a patient with follicular small cleaved cell lymphoma. At the same time, an Epstein-Barr virus (EBV) nuclear antigen (EBNA)-positive cell line, FL-18-EB, was established from the EBV-infected culture of the same pleural effusion cells. Both cell lines had the same monoclonal surface immunoglobulin (IgG kappa), and they had the same karyotype as that of the fresh pleural effusion cells in which a reciprocal translocation between the long arm of chromosomes 14 and 18 [t(14;18)(q32;q21)] was detected. Gene rearrangement analysis of immunoglobulin heavy-chain gene (JH) and kappa light-chain gene (J kappa) showed the same rearranged configuration in the two cell lines; however, some morphological and phenotypic differences were found. The FL-18-EB cells, which were morphologically similar to common EBNA-positive lymphoblastoid cell lines of normal B cell origin at the initial phase of culture, were larger than the FL-18 cells and contained multinucleated giant cells. The FL-18 cells lacked cytoplasmic immunoglobulin and were positive for common acute lymphoblastic leukemia antigen (CALLA), whereas the FL-18-EB cells had cytoplasmic immunoglobulin and were negative for CALLA. Thus, the phenotype of FL-18-EB seems to be a result of a shift by EBV infection to a more mature stage in the B cell differentiation pathway than that of FL-18. The paired availability of EBV-free and EBV-infected cell lines of a neoplastic clone is unique and valuable in considering EBV infectibility of neoplastic B cells and resultant phenotypic changes.

Phenotype versus immunoglobulin and T‐cell receptor genotype of Hodgkin‐derived cell lines: Activation of immature lymphoid cells in Hodgkin's disease

Three Hodgkin-derived cell lines (L428, L540, and CO) were studied for rearrangements and expression of immunoglobulin and T-cell receptor genes, and their genotype was compared to the phenotype. As far as the genotype is concerned, all 3 cell lines have characteristics of lymphoid cells; L428 of B, and L540 and CO of T-cell origin. L428 cells have one Ig heavy chain allele rearranged to C gamma and transcribed into RNA, while the second is deleted. Furthermore, L428 cells show an unusual immunoglobulin kappa light chain gene rearrangement involving deletion of the kappa constant gene in one allele, while the remaining kappa and lambda loci are in germline configuration. L540 and CO have, in contrast to L428 cells, the immunoglobulin genes in germline and T-cell receptor genes rearranged. The T-cell receptor beta and gamma genes are rearranged in both L540 and CO, whereas a rearrangement in the alpha locus was detected in L540 cells only. RNA of the size of functional beta chain transcripts was found in CO cells and of the size of functional alpha chain transcripts in L540 cells. All 3 cell lines are classified as immature lymphoid cells with respect to the limited expression of B- and T-cell antigens, respectively, and to the incomplete expression of their antigen receptor. The immaturity of lymphoid differentiation contrasts with the expression of activation antigens, i.e. Ki-1, Ki-24, HLA-DR, and IL-2 receptor. The immaturity of the cells excludes the possibility that the cells were activated along the physiological pathway, i.e. by interaction of the cell with antigen. The results obtained on the cell lines are in accordance with in vivo studies and suggest that Hodgkin and Sternberg-Reed cells are immature lymphoid cells which are activated by a still unknown mechanism.

Protein-Binding Sites in Ig Gene Enhancers Determine Transcriptional Activity and Inducibility

Individual protein-binding sites within the mouse immunoglobulin heavy chain and kappa light chain gene enhancers were altered, making it possible to examine the functional role of the sites during transcription. The E motifs, which bind factors that are present in many if not all cells, mostly behave as transcriptional activating sites. The only known heavy chain enhancer site that binds a lymphocyte-specific factor, the "octamer" site, plays a critical role in transcription but only in a truncated form of the enhancer. In the full enhancer, no one site is crucial because of an apparent functional redundancy. The site in the kappa enhancer that binds a factor specific to mature B cells, kappa B, was crucial to the constitutive activity of the enhancer in B cells. This factor is also inducible in pre-B cells, and the site was necessary for inducibility of the kappa enhancer. Thus, the sites defined by protein binding are important for the functional activity of immunoglobulin enhancers, with the sites that bind proteins restricted in their cellular distribution playing the most important roles.

Rearrangement and expression of rabbit immunoglobulin kappa light chain gene in transgenic mice.

To determine whether a foreign unrearranged immunoglobulin gene can be functionally rearranged and expressed in vivo, a rabbit b9 kappa light chain gene construct containing a single germ-line kappa chain variable (V) region gene (V kappa), the five kappa chain joining (J) segments (J kappa), and the kappa chain constant (C) region germ-line gene (C kappa) was introduced into fertilized mouse eggs. Eleven transgenic mice carrying 1-30 copies of the rabbit kappa gene construct were obtained. Rearrangement of the transgene due to V kappa-J kappa recombination was observed in the spleen of all the mice lines analyzed. Only the J kappa 1 and J kappa 2 segments, which have canonical sequences for rearrangement and high-level expression, are utilized in assembly of the rabbit gene. V kappa-J kappa 1 and V kappa-J kappa 2 joining was also observed in the thymus but not in nonlymphoid tissue. Polyadenylylated rabbit kappa chain transcripts of 1.2 kilobases were found in the spleen of the transgenic mice. The level of transcription was low despite a high level of rearrangement. Three transgenic mice lines secreted kappa light chains encoded by the foreign rabbit gene. Serum rabbit kappa chains were associated with mouse mu and gamma 1 heavy chains. However, hybrid antibody molecules containing both rabbit and mouse kappa light chains were also found in the serum of these animals. These results suggest that, although the rabbit kappa chain gene construct contains the necessary sequences for gene assembly, sequences implicated in stage- and tissue-specific regulation of kappa chain gene rearrangement are either missing or not recognized by mouse lymphoid cells.

Cell-type-specific transcription of an immunoglobulin kappa light chain gene in vitro.

We have established a cell-free system, derived from a human B-cell lymphoma, in which immunoglobulin kappa light chain gene promoters are both accurately transcribed and regulated in a cell-type-specific manner. Thus, accurate transcription from the T1 kappa light chain gene promoter was much more efficient in B-cell extracts than in HeLa cell extracts, whereas control promoters (adenovirus major late and histone H2B) were transcribed equally well in either extract. More important, the increased kappa light chain gene transcription in B-cell extracts was dependent upon upstream sequences (containing the conserved decanucleotide element) previously shown to be necessary for B-cell-specific transcription in vivo; in contrast, removal of these sequences had no effect on the low level of kappa transcription in HeLa extracts. The maximal level of upstream sequence-mediated transcription was dependent upon template topology. These studies show that there is at least one B-cell-specific factor that stimulates transcription from purified DNA templates, and they further suggest that the in vivo action of the factor(s) on other components of the transcription machinery is direct rather than indirect (e.g., via the maintenance of an open chromatin structure). The cell-free system described here should facilitate both purification and functional studies of the B-cell-specific factor(s).

Cell-type preference of immunoglobulin kappa and lambda gene promoters.

Immunoglobulin gene constant regions are known to be associated with strictly tissue-specific enhancer elements. Until recently the promoter of the variable region, which becomes linked to the constant region by somatic rearrangement, could have been viewed as a passive recipient of the enhancer stimulus. Here we show that the promoters of the immunoglobulin kappa and lambda light chain genes are approximately 20-30 times more active in lymphoid cells than in non-lymphoid cells. To avoid the problem of differential mRNA stability upon transfection of immunoglobulin genes into non-lymphoid cells we have constructed chimeric genes. All kappa mRNA sequences were progressively deleted to fuse the kappa gene promoter to a globin gene coding body. A similar chimeric gene was constructed with the promoter of the lambda gene. The cell-type preference of the promoter may be exploited during B-lymphocyte differentiation to regulate the immunoglobulin gene promoter independently from the enhancer.

Gm and Km allotypes in rheumatoid arthritis.

Immunoglobulin heavy chain (Gm) and kappa light chain (Km) allotype and phenotype frequencies were compared in 173 patients with rheumatoid arthritis (RA) and in 798 controls. No significant differences were found between allotype or phenotype frequencies in overall RA and control groups. However, the Gm(zaxfngb) phenotype and G1m(x) allotype were increased in HLA-DR4 positive RA patients compared with DR4 negative patients and controls, suggesting that immunoglobulin heavy chain genes interact with HLA in the pathogenesis of RA. All four patients with pulmonary fibrosis were Km(1) positive suggesting a possible role for immunoglobulin kappa light chain genes in the pathogenesis of pulmonary fibrosis found in rheumatoid patients.

IL3-dependent mouse clones that express B-220 surface antigen, contain ig genes in germ-line configuration, and generate B lymphocytes in vivo

The continuously proliferating clones L/B AgA2, CB/Bm 7, Ba/C1, and Bc/Bm 11 were established from bone marrow of MRL/LPR, CBA/J, and BALB/c mice. These clones carry the B cell lineage surface antigen B-220 but not antigens normally expressed on mature B lymphocytes, myeloid cells, or T lymphocytes. Their immunoglobulin mu heavy chain and kappa light chain genes are in germ-line configuration. The G418 resistance gene was introduced into each clone with a retrovirus vector and then used as a selective marker for the progeny of transfected cells. Clones L/B AgA2, CB/Bm 7, and Bc/Bm 11, but not Ba/C1, could develop into antibody-secreting cells after in vivo transfer. None gave rise to cells responsive to polyclonal T cell activators, nor did any differentiate into cells that could develop into granulocyte/macrophage-colony-forming cells in vitro. All grew in interleukin 3 but not in other cytokines. We conclude that clones L/B AgA2, CB/Bm 7, and Bc/Bm 11 are early precursors of B lymphocytes.