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1.
J Mol Biol ; 292(2): 217-27, 1999 Sep 17.
Article in English | MEDLINE | ID: mdl-10493870

ABSTRACT

The terminal deoxynucleotidyl transferase gene (TdT) is expressed in mice only in early B and T lymphoid precursors a few days after birth. Transactivating factors have been shown to contribute to the lymphoid specific expression of TdT, but they do not account entirely for the restriction of its expression to early precursors. Since tissue-specific expression can be modulated by other mechanisms such as DNA methylation and DNA accessibility, we evaluated the methylation pattern of the TdT gene in various expressing and non-expressing tissues and cell lines. Lymphoid and non-lymphoid organs differed significantly in their methylation profiles. In the thymus nearly complete demethylation of a Hha I site in the promoter was associated with high levels of TdT transcription. There was similar, but weaker demethylation of the TdT promoter in bone marrow, possibly due to the presence of a few TdT expressing B cell precursors. The same methylation status was also associated with TdT expression in different B and T cell lines. Kinetic studies of TdT gene demethylation and TdT transcription during thymus development showed that changes in methylation status were also involved in the differential expression of TdT in fetal and adult life. Footprinting experiments revealed the existence of three regions specifically protected by nuclear extracts from TdT -expressing cells. Together, these results suggest that promoter demethylation is involved in the control of TdT expression and implicate new promoter regions in this regulation.


Subject(s)
DNA Methylation , DNA Nucleotidylexotransferase/genetics , Promoter Regions, Genetic/genetics , Age Factors , Animals , B-Lymphocytes/metabolism , Blotting, Southern , Bone Marrow/metabolism , Cell Line , DNA Footprinting , Gene Expression Regulation, Developmental , Kinetics , Mice , Mice, Inbred C57BL , T-Lymphocytes/metabolism , Thymus Gland/metabolism
2.
J Immunol ; 162(4): 2123-8, 1999 Feb 15.
Article in English | MEDLINE | ID: mdl-9973486

ABSTRACT

Nontemplated (N) nucleotide additions contribute significantly to the junctional diversity of all Ag receptor chains in adult mice except Ig light (L) chains, primarily because terminal deoxynucleotidyl transferase (TdT) expression is turned off at the time of their rearrangement in pre-B cells. However, because some Ig L chain gene rearrangements are detectable earlier during B cell ontogeny when TdT expression is thought to be maximal, we have examined the junctional processing of kappa- and lambda-chain genes of CD45(B220)+CD43+ pro-B cells from mu MT mice. We found that both kappa and lambda coding junctions formed in these B cell precursors were extensively diversified with N-region additions. Together, these findings demonstrate that Ig L chain genes are equally accessible to TdT in pro-B cells as Ig heavy chain genes. Surprisingly, however, the two L chain isotypes differed in the pattern of N addition, which was more prevalent at the lambda-chain locus. We observed the same diversity pattern in pre-B cells from TdT-transgenic mice. These results suggest that some aspects of TdT processing could be influenced by factors intrinsic to the sequence of Ig genes and/or the process of V(D)J recombination itself.


Subject(s)
B-Lymphocytes/metabolism , Gene Rearrangement, B-Lymphocyte, Light Chain , Immunoglobulin J-Chains/genetics , Immunoglobulin kappa-Chains/genetics , Immunoglobulin lambda-Chains/genetics , Immunoglobulin mu-Chains/genetics , Stem Cells/metabolism , Animals , B-Lymphocytes/enzymology , Cell Differentiation/genetics , Cell Differentiation/immunology , DNA Nucleotidylexotransferase/biosynthesis , Female , Immunoglobulin Isotypes/chemistry , Immunoglobulin Isotypes/genetics , Immunoglobulin J-Chains/chemistry , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/genetics , Immunoglobulin kappa-Chains/chemistry , Immunoglobulin lambda-Chains/chemistry , Immunoglobulin mu-Chains/chemistry , Lymphopenia/enzymology , Lymphopenia/genetics , Lymphopenia/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Reading Frames/immunology , Stem Cells/enzymology
3.
J Immunol ; 161(12): 6657-63, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9862694

ABSTRACT

During neonatal life, Ig diversity is limited in many respects. The absence of terminal deoxynucleotidyl transferase (TdT) expression with the consequent lack of nontemplated addition during the neonatal period, coupled with the predominant usage of a single D(H) reading frame (RF), leads to severe limitations of diversity in the CDR3 region of Ig heavy (H) chains. The neonatal Ig H chain repertoire is also characterized by restricted V(H) usage, with predominant expression of certain V(H) segments, such as V(H)81x, that are rarely evident during adult life. In this report, we examine the effect of enforced TdT expression on the neonatal repertoire of V(H)81xDJ(H) rearrangements. We find that TdT synthesis abrogates D(H) RF bias during the fetal/neonatal period through a Ig-receptor-independent mechanism. These findings suggest that D(H) RF bias during neonatal life is determined largely by homology-directed joining. We also find that TdT synthesis alters the selection of productively rearranged V(H)81xDJ(H) alleles in the neonatal spleen through a Ig-receptor-dependent mechanism. Analysis of predicted CDR3 amino acid sequences indicates that positive selection of V(H)81x-encoded H chains is correlated with the presence of a consensus sequence immediately adjacent to the V(H) segment. These data support the hypothesis that the CDR3 region is critical in determining the ability of V(H)81x-encoded H chains to form functional receptors that support positive selection of B lymphocytes. Together, our results demonstrate that TdT can indirectly influence the Ig repertoire by influencing both receptor-dependent and receptor-independent selection processes.


Subject(s)
Antibody Diversity , B-Lymphocytes/immunology , DNA Nucleotidylexotransferase/biosynthesis , Gene Rearrangement, delta-Chain T-Cell Antigen Receptor , Immune System/embryology , Immunoglobulin delta-Chains/genetics , Amino Acid Sequence , Animals , Animals, Newborn , B-Lymphocytes/metabolism , Clonal Deletion , Consensus Sequence , DNA Nucleotidylexotransferase/genetics , Enzyme Induction , Genes, Immunoglobulin , Immune System/growth & development , Mice , Mice, Transgenic , Models, Immunological , Molecular Sequence Data , Open Reading Frames
4.
J Mol Biol ; 281(5): 815-25, 1998 Sep 04.
Article in English | MEDLINE | ID: mdl-9719637

ABSTRACT

The hallmark of Fanconi anemia (FA), a rare inherited cancer prone disorder, is a high level of chromosome breakage, spontaneous and induced by cross-linking agents. The increased genomic instability of FA is reflected at the gene level by an overproduction of intragenic deletions. Two of the eight FA genes have been cloned, however, their function remains unknown. We recently demonstrated that the lack of functional FA genes lead to a marked decrease in the fidelity of non-homologous end-joining, a pathway that mammalian cells predominantly use to repair DNA double-strand breaks (DSB). Knowing that specific DSB are generated during V(D)J recombination, here we have examined the molecular features of V(D)J rearrangements in normal and FA lymphoblasts belonging to complementation groups C and D. Using appropriate extrachromosomal recombination substrates, V(D)J coding and signal joint formation have been analysed quantitatively and qualitatively. Our results show that the frequency of coding and signal joint formation was not significantly different in normal and FA cells. However, when the fidelity of the V(D)J reaction was examined, we found that in normal human lymphoblasts V(D)J recombination proceeds with high precision, whereas, in FA cells a several fold increase in the frequency of aberrant rearrangements is associated with V(D)J coding joint formation. The abnormal recombinants that we recovered in FA are consistent with excessive degradation of DNA ends generated during the V(D)J reaction. On the basis of these findings, we propose a working model in which FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion proneness.


Subject(s)
Fanconi Anemia/genetics , Gene Rearrangement/genetics , Genes, Immunoglobulin/genetics , Recombination, Genetic/genetics , Cell Line , Chromosome Breakage/genetics , Humans , Plasmids/genetics , Sequence Analysis, DNA , Transfection/genetics
5.
J Immunol ; 160(9): 4254-61, 1998 May 01.
Article in English | MEDLINE | ID: mdl-9574527

ABSTRACT

We analyzed the progeny of individual multipotent hemopoietic cells, derived from the para-aortic splanchopleura, the earliest identified source of lymphocyte precursors in pre-liver mouse embryos. Single precursors were expanded in an in vitro culture system that permits both commitment and differentiation of B cell precursors. We show that from one single multipotent progenitor we could obtain large numbers of B cell precursors that rearrange the Ig heavy chain genes and generate a repertoire as diverse as that observed in adult populations. N region additions are present at V(D)J junctions, showing that terminal deoxynucleotidyl transferase expression has been switched on and is not, consequently, an intrinsic property of adult stem cells. Throughout the culture period, cells show a majority of DJ vs V(D)J rearrangements and a ratio of 2:1 of nonproductive to productive V(D)J rearrangements, which is close to the expected frequency in the absence of selection. In addition, counterselection for D-J rearrangements in reading frame 2 is observed in V(D)J joints, and allelic exclusion was consistently observed. We conclude that of the three events associated with heavy chain rearrangement, two of them, namely allelic exclusion and counterselection of cells in which the D segment is in reading frame 2, are intrinsic to the cell, while selection of productive heavy chain rearrangements is induced in the bone marrow environment.


Subject(s)
B-Lymphocytes/immunology , Gene Rearrangement, B-Lymphocyte , Genes, Immunoglobulin , Hematopoietic Stem Cells/immunology , Immunoglobulin Heavy Chains/genetics , Animals , B-Lymphocytes/cytology , Cell Differentiation/immunology , Hematopoietic Stem Cells/cytology , Immunoglobulin Heavy Chains/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
6.
Cancer Res ; 57(20): 4600-7, 1997 Oct 15.
Article in English | MEDLINE | ID: mdl-9377575

ABSTRACT

The 180BR cell line was derived from an acute lymphoblastic leukemia patient who overresponded to radiation therapy and died following radiation morbidity. 180BR cells are hypersensitive to the lethal effects of ionizing radiation and are defective in the repair of DNA double-strand breaks (DSBs). The levels and activity of the proteins of the DNA-dependent protein kinase complex are normal in 180BR cells. To facilitate a measurement of V(D)J recombination, we have characterized 180BRM, a SV40-transformed line derived from 180BR. 180BRM retains the radiosensitivity and defect in DSB repair characteristic of 180BR. The activities associated with DNA-dependent protein kinase are also normal in 180BRM cells. The ability to carry out V(D)J recombination is comparable in 180BRM and a reference control transformed human cell line, MRC5V1. These results show that 180BR and 180BRM differ from the rodent mutants belonging to ionizing radiation complementation groups 4, 5, 6, and 7 and, therefore, represent a new mutant phenotype, in which a defect in DNA DSB rejoining is not associated with defective V(D)J recombination. Furthermore, we have shown that 180BR can arrest at the G1-S and G2-M cell cycle checkpoints after irradiation. These results confirm that 180BR can be distinguished from ataxia telangiectasia.


Subject(s)
Cell Survival/radiation effects , DNA Damage , DNA-Binding Proteins , Protein Serine-Threonine Kinases/metabolism , Radiation Tolerance/genetics , Cell Cycle/genetics , Cell Line, Transformed , Cell Nucleus/metabolism , Cobalt Radioisotopes , DNA Nucleotidyltransferases/metabolism , DNA-Activated Protein Kinase , Dose-Response Relationship, Radiation , Fibroblasts , Gamma Rays , Genetic Complementation Test , Humans , Kinetics , Nuclear Proteins , Phenotype , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/radiotherapy , Recombination, Genetic , Tumor Cells, Cultured , VDJ Recombinases
7.
J Immunol ; 158(2): 715-23, 1997 Jan 15.
Article in English | MEDLINE | ID: mdl-8992987

ABSTRACT

N region diversity in Ag receptors is a developmentally regulated process in B and T cells that correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). Absent in fetal and newborn mice, TdT expression is restricted to early T and pro-B cells in adults. To extend the TdT expression pattern throughout B cell ontogenesis, we generated transgenic mice carrying a TdT cDNA under the regulatory elements of the N-myc gene and the IgH enhancer. High expression was observed in secondary lymphoid organs consistent with TdT activity beyond the pre-B cell stage. This suggests that TdT transgene expression is not down-regulated as is the endogenous gene. Unlike normal mice, extensive N region diversity was found in rearranged lambda light chain genes of adult transgenic animals. Therefore, expression of TdT appears sufficient for N region diversity to occur at any Ig locus. More importantly, expression of the transgene takes place during fetal development. As a consequence, the potential fetal B cell repertoire is modified as both rearranged heavy and light chain genes now show N region additions. Constitutive expression of TdT throughout B cell differentiation does not therefore appear deleterious and suggests that TdT is recruited only to participate in the V(D)J recombination process.


Subject(s)
Antibody Diversity/genetics , B-Lymphocytes/immunology , DNA Nucleotidylexotransferase/biosynthesis , Gene Rearrangement/immunology , Immunoglobulin Variable Region/genetics , Animals , B-Lymphocytes/enzymology , Base Composition , Cell Differentiation/genetics , Cell Differentiation/immunology , Immunoglobulin Light Chains/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Sequence Homology, Nucleic Acid , Transfection
9.
EMBO J ; 14(17): 4221-9, 1995 Sep 01.
Article in English | MEDLINE | ID: mdl-7556063

ABSTRACT

Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy-terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site-specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3' end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half-life of the TdTL protein in these cells is 2-fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions.


Subject(s)
Alternative Splicing , Bone Marrow/enzymology , DNA Nucleotidylexotransferase/metabolism , Isoenzymes/metabolism , Thymus Gland/enzymology , Animals , B-Lymphocytes/enzymology , Base Sequence , Cell Line , Chlorocebus aethiops , DNA Nucleotidylexotransferase/biosynthesis , DNA Primers , Hematopoietic Stem Cells/enzymology , Isoenzymes/biosynthesis , Kinetics , Mice , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Recombinant Proteins/metabolism , Subcellular Fractions/enzymology , Transfection
10.
Res Immunol ; 146(6): 373-82, 1995.
Article in English | MEDLINE | ID: mdl-8719661

ABSTRACT

A high-affinity anti-tenanus toxoid (TT) human monoclonal antibody showing neutralizing activity was isolated from a fusion between mouse myeloma and human splenic cells. Fab fragments from this antibody were obtained using a recombinant phage surface-display expression system. The parental antibody and the corresponding Fab had identical immunological activities, including specificity and affinity. These results confirm the feasibility of developing Escherichia coli expression of monoclonal human Fab from hybridoma cells.


Subject(s)
Antibodies, Monoclonal/metabolism , Immunoglobulin Fab Fragments/metabolism , Tetanus Toxoid/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Base Sequence , Coliphages/genetics , DNA Primers/genetics , Escherichia coli/genetics , Gene Library , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , In Vitro Techniques , Mice , Molecular Sequence Data , Neutralization Tests , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism
11.
Eur J Immunol ; 23(8): 1789-95, 1993 Aug.
Article in English | MEDLINE | ID: mdl-8344341

ABSTRACT

We have previously shown that unlike endogenous chi genes, unrearranged chi transgenes undergo V chi-J chi recombination in Tas well as B cells of transgenic mice. To determine whether the difference in recombination specificity of the transgenic and endogenous chi genes is associated with differences in DNA structure, the methylation status of the endogenous genes and three unrearranged chi transgenes was compared. The J chi-C chi locus of the transgenes was found to be hypomethylated in all tissues of the transgenic mice. In contrast, methylation of the endogenous chi genes was tissue and developmentally regulated. Hypomethylation of the endogenous J chi-C chi region occurs only in cells of the B lineage undergoing, or having completed chi gene recombination. Transfection of fibroblasts from transgenic and control mice with the recombination activating genes, Rag1 and Rag2, led to a high level of rearrangement of the hypomethylated transgenic, but not the endogenous chi genes. These results suggest that hypomethylation defines an accessible state of the chi locus and that methylation/demethylation could be involved in the control of chi gene rearrangement during lymphocyte differentiation.


Subject(s)
DNA-Binding Proteins , Gene Rearrangement , Genes, Immunoglobulin , Homeodomain Proteins , Immunoglobulin kappa-Chains/genetics , Animals , Base Sequence , Cell Line , Female , Immunoglobulin Constant Regions/genetics , Immunoglobulin Joining Region/genetics , Male , Methylation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Proteins/genetics
12.
Nucleic Acids Res ; 21(5): 1187-91, 1993 Mar 11.
Article in English | MEDLINE | ID: mdl-8464703

ABSTRACT

A new form of TdT mRNA has been identified by screening a mouse thymus cDNA library. It contains an open reading frame of 1527 base pairs corresponding to a protein containing 509 aminoacids, whereas the previously identified mouse TdT mRNA is composed of 1587 base pairs and encodes a protein of 529 aminoacids. Analysis of a mouse genomic clone containing the 3' portion of the TdT gene shows that these twenty additional aminoacids are encoded by an additional exon located between exons X and XI. Both forms of TdT mRNA are present in the thymus and could be generated by alternative splicing. The cDNA reported here corresponds to the major form of TdT mRNA in Balb/c mice and closely resembles human and bovine TdT cDNA. Expression of this cDNA in mammalian cells shows that it encodes a functional protein capable of catalysing N region insertions at the recombination junction of an episomic recombination substrate.


Subject(s)
DNA Nucleotidylexotransferase/genetics , RNA Splicing , Thymus Gland/enzymology , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , DNA , DNA Nucleotidylexotransferase/metabolism , Humans , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Nucleotides/metabolism , Restriction Mapping
13.
Proc Natl Acad Sci U S A ; 89(7): 2799-803, 1992 Apr 01.
Article in English | MEDLINE | ID: mdl-1557386

ABSTRACT

The somatic diversity immunoglobulin and T-cell receptor diversity is largely provided by the junctional variation created during site-specific rearrangement of separately encoded gene segments. Using a transient transfection assay, we demonstrate that the recombination activating genes Rag1 and Rag2 direct site-specific rearrangement on an artificial substrate in poorly differentiated as well as in differentiated nonlymphoid cell lines. In addition to a high frequency of precise recombination events, coding joints show deletions and more rarely P-nucleotide insertions, reminiscent of immunoglobulin and T-cell receptor junctions found in fetal tissues. N-region insertions, which are characteristic of adult junctional diversity, are obtained at high frequency upon transfection of a terminal deoxynucleotidyltransferase expression vector together with Rag1 and Rag2. These results show that only three lymphoid-specific factors are needed to generate all types of junctional diversity observed during lymphoid development.


Subject(s)
DNA Nucleotidylexotransferase/genetics , DNA-Binding Proteins , Gene Rearrangement, B-Lymphocyte , Gene Rearrangement, T-Lymphocyte , Genes, Immunoglobulin , Homeodomain Proteins , Proteins/physiology , Receptors, Antigen, T-Cell/genetics , Recombination, Genetic , 3T3 Cells , Animals , Mice , Transfection
14.
Eur J Immunol ; 22(2): 381-5, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1537376

ABSTRACT

Mice immunized with the recombinant antigen 11.1 beta-galactosidase, consisting of 22 repeats of the nine-amino acid unit from Plasmodium falciparum antigen 11.1, produced antibodies reacting with human serum albumin. A positive reaction was observed in dot-blot assays, in enzyme-linked immunosorbent assay and on immunoblots of sodium dodecyl sulfate polyacrylamide gels as well as two-dimensional gels. Binding was specific for human albumin, as no reaction could be detected on bovine serum albumin, hen egg ovalbumin, rat serum albumin or another abundant human serum protein, the alpha 2-macroglobulin. In addition, rabbit antibodies raised to human serum albumin reacted with keyhole lympet hemocyanin coupled to synthetic dimers of the nine-amino acid repeats of the P. falciparum 11.1 antigen. These data indicate antigenic relationship between the 11.1 antigen and human albumin. The proteins have a short sequence of homology in a region where human serum albumin differs from the albumins of other species.


Subject(s)
Antibodies, Protozoan/immunology , Antigens, Protozoan/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Serum Albumin/immunology , Amino Acid Sequence , Animals , Antigens, Protozoan/chemistry , Cross Reactions , Humans , Molecular Sequence Data , Peptides/immunology , Protozoan Proteins/chemistry , Serum Albumin/chemistry
15.
Nucleic Acids Res ; 19(19): 5339-44, 1991 Oct 11.
Article in English | MEDLINE | ID: mdl-1923817

ABSTRACT

The mouse heavy chain immunoglobulin promoter VH441 can lead in vitro to bidirectional transcription, due to a symmetrical organization of immunoglobulin heavy chain promoters with two TATA-like sequences bracketing the upstream promoter element ATGCAAAT (the so called octamer). We demonstrate here that divergent transcription also occurs in vivo in mature B cells from a myeloma which expresses the VH441 gene and even from the spleen of BALB/c mice. The level of VH441 divergent transcript increases in the spleen of BALB/c mice after immunisation by beta-(1,6)-galactan, showing that it is expressed in B cells which actively transcribe the VH441 gene. The divergent transcript has been characterized: its major transcription start site was mapped within 33 base pairs from the divergent TATA-like region, it is unspliced and not polyadenylated. In the light of these results, the functions of the divergent transcript and the bidirectional promoter are discussed.


Subject(s)
B-Lymphocytes/metabolism , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Multiple Myeloma/genetics , Promoter Regions, Genetic/genetics , Animals , B-Lymphocytes/drug effects , Base Sequence , Blotting, Northern , Galactans/pharmacology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Spleen/metabolism , TATA Box/genetics , Transcription, Genetic/physiology , Tumor Cells, Cultured
16.
Mol Immunol ; 27(5): 429-33, 1990 May.
Article in English | MEDLINE | ID: mdl-1973259

ABSTRACT

Antibody E225 reacts with a private idiotope of the anti-lysozyme antibody D1.3. A complex between the Fab fragments from these BALB/c monoclonal antibodies has been crystallized and the determination of the three-dimensional structure of this idiotope-anti-idiotope complex is under way. The nucleotide VH and VL sequences of E225 presented here have been determined to provide the amino acid sequence information necessary for the interpretation of the high resolution electron density maps of the complex, obtained by X-ray crystallography. The cDNAs synthesized from the Vkappa and VH mRNAs were cloned in E. coli. Both cDNA strands were sequenced by the dideoxy termination method. The translated amino acid sequence shows that Vkappa, VH correspond to groups five (V) and II(b) of mouse immunoglobulin light and heavy chains, respectively. Sequence alignments between the complementarity determining regions of E225 and the antigenic determinant of lysozyme recognized by D1.3 do not indicate whether or not the anti-idiotopic antibody structurally mimics the external antigen.


Subject(s)
Antibodies, Anti-Idiotypic/genetics , Immunoglobulin Variable Region/genetics , Muramidase/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Base Sequence , Cloning, Molecular , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Poly A/genetics , RNA, Messenger/genetics
17.
Nucleic Acids Res ; 17(12): 4745-55, 1989 Jun 26.
Article in English | MEDLINE | ID: mdl-2501761

ABSTRACT

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.


Subject(s)
Enhancer Elements, Genetic , Immunoglobulin kappa-Chains/genetics , Plasmacytoma/genetics , Animals , Base Sequence , Chimera , DNA-Binding Proteins/genetics , Immunoglobulin kappa-Chains/metabolism , Introns , Mice , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Plasmacytoma/immunology , Plasmacytoma/metabolism , Rabbits , Transfection , Tumor Cells, Cultured
18.
Nucleic Acids Res ; 17(5): 1977-87, 1989 Mar 11.
Article in English | MEDLINE | ID: mdl-2494644

ABSTRACT

We show that the promoter from the mouse VH441 heavy-chain immunoglobulin gene, when present on plasmids transiently introduced into myeloma cells, promotes transcription bidirectionally, due to the presence on both strands of TATA-like sequences bracketing the highly conserved decanucleotide element. The two divergent promoters compete for the transcriptional machinery, their relative strength ultimately reflecting the likeness of the two TATA boxes to the consensus sequence. Moreover, their relative activity is also strongly influenced by certain point mutations within the distally located heavy-chain enhancer. The bearing of these results on current concepts of promoter function is discussed.


Subject(s)
Genes, Immunoglobulin , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Animals , Base Sequence , Binding, Competitive , Cell Line , Enhancer Elements, Genetic , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Mice , Molecular Sequence Data , Mutation , Plasmacytoma/genetics , Transcription, Genetic
19.
FEBS Lett ; 222(2): 337-40, 1987 Oct 05.
Article in English | MEDLINE | ID: mdl-2820808

ABSTRACT

A mouse renin-1 gene promoter fragment, normally inactive in B-cells, becomes a potent promoter in these cells after insertion of the highly conserved decanucleotide (dc/cd sequence) of immunoglobulin heavy and light chain promoters [(1987) EMBO J. 6, 1685-1690]. We observe retarded complexes of the same electrophoretic mobility when the cd-containing renin promoter fragment or an authentic immunoglobulin heavy chain promoter fragment is incubated with a nuclear extract from myeloma cells, suggesting that the renin promoter is activated due to its acquired ability to bind a B-cell-specific positive factor. No retarded complexes are observed with the original renin promoter fragment thus questioning the presence of a repressor as an explanation for its lack of activity in B-cells.


Subject(s)
B-Lymphocytes/immunology , Immunoglobulin Heavy Chains/genetics , Promoter Regions, Genetic , Renin/genetics , Animals , Binding Sites , DNA Transposable Elements , Gene Expression Regulation , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Light Chains/genetics , Mice , Multiple Myeloma/genetics , Multiple Myeloma/immunology , Renin/metabolism
20.
EMBO J ; 6(6): 1685-90, 1987 Jun.
Article in English | MEDLINE | ID: mdl-3111845

ABSTRACT

A conserved decanucleotide (ATGCAAATNA) is present 45-60 nucleotides upstream from the transcription startpoint in all immunoglobulin heavy chain promoters (VH promoters). We have introduced this decanucleotide (cd sequence) at a similar position into the upstream flanking sequence of the mouse Renin-1 gene. This gene is only transcribed in highly specialized tissues, and the fragment used here (-449 to +30 with respect to the main transcription startpoint) has little promoter activity in fibroblastic or myeloma cell lines, even if coupled to a functional enhancer. In contrast, after insertion of the decanucleotide, this fragment, while still inactive in non-lymphoid cells, becomes a potent promoter in B-cells when associated with SV40 or immunoglobulin heavy chain enhancer. In all respects, the engineered fragment behaves like an authentic VH promoter isolated in this laboratory, except that it is even more active in B-cells. Deletion experiments show that all renin sequences are dispensable for the activity of the chimaeric promoter, except probably for the renin TATA box which defines the precise transcription startpoint. We conclude that the decanucleotide is sufficient to activate a promoter in B-cells but not in non-B-cells, and therefore that no other element is needed to account for the B-cell specificity of the VH promoter. In addition, our results suggest that the lack of activity of the renin promoter in non cognate cells is not due to the binding of a repressor.


Subject(s)
B-Lymphocytes/immunology , Genes , Immunoglobulin Heavy Chains/genetics , Promoter Regions, Genetic , Transcription, Genetic , Animals , Base Sequence , Cell Line , Cells, Cultured , Enhancer Elements, Genetic , Mice , Plasmacytoma/immunology , Renin/genetics
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