Mouse Immunoglobulin Heavy Chain Gene Research Articles

The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system

Activation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by somatic hypermutations and class switch recombination and is supposed to deaminate cytidines in DNA, while its homolog APOBEC-1 edits apolipoprotein (apo) B mRNA by cytidine deamination. We studied the editing activity of APOBEC-1 and AID in yeast using the selectable marker Gal4 linked to its specific inhibitor protein Gal80 via an apo B cassette (Gal4-C) or via the variable region of a mouse immunoglobulin heavy chain gene (Gal4-VH). Expression of APOBEC-1 induced C to U editing in up to 15% of the Gal4-C transcripts, while AID was inactive in this reaction even in the presence of the APOBEC-1 complementation factor. After expression of APOBEC-1 as well as AID approximately 10 −3 of yeast cells survived low stringency selection and expressed β-galactosidase. Neither AID nor APOBEC-1 mutated the VH sequence of Gal4-VH, and consequently the yeast colonies did not escape high stringent selection. AID, however, induced frequent plasmid recombinations that were only rarely observed with APOBEC-1. In conclusion, AID cannot substitute APOBEC-1 to edit the apo B mRNA, and the expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed Gal4-VH sequence. Cofactors for AID induced somatic hypermutations of immunoglobulin variable regions, that are present in B cells and a variety of non-B cells, appear to be missing in yeast. In contrast to APOBEC-1, AID alone does not exhibit an intrinsic specificity for its target sequences.

The absence of DNA polymerase κ does not affect somatic hypermutation of the mouse immunoglobulin heavy chain gene

During the immune response to T cell-dependent antigen, somatic hypermutation (SHM) is introduced into immunoglobulin (Ig) genes. The variable region is the target for SHM and it is here that DNA lesions are introduced and mutations are generated. It has been suggested that error-prone DNA polymerase(s) may play an important role in this mutagenesis phase. Recently, DNA polymerase κ (Polκ), which belongs to the Y-family of DNA polymerases, was identified. Since a hot spot of SHMs (RGYW motif) is also a hot spot of mutations by human Polκ, this enzyme was suggested to be an SHM instigator. In order to address the question whether Polκ is involved in SHM, we immunized Polκ-deficient mice and analyzed the SHM of the Ig heavy chain gene. We found that the SHM frequency and spectrum were indistinguishable between the Polκ knockout mice and control mice. These results suggested that Polκ is not essential for this process.

Analysis of clonal diversity in mouse immunoglobulin heavy chain genes selected for size of the antigen combining site

Size-diversity of lg and T cell receptor antigen binding (CDR3) regions can be visualized by “CDR3 fingerprinting”, and provides an estimate of B- or T-cell repertoire complexity. The method does not identify clonal diversity, however, which can only be determined by random sequencing of the CDR3s. In this study we demonstrate that a combination of fingerprinting and single strand conformation polymorphism (SSCP) analysis can be used for a rapid estimatation of clonal diversity within mouse lg antigen binding regions selected for size. This application may be useful in the analysis of clonal expansion within B- and T-cell repertoires.

Effect of transcriptional regulatory sequences on autonomous replication of plasmids in transient mammalian systems.

Transcriptional regulatory sequences often influence the efficiency of DNA replication, directly or indirectly, in bacteria, yeast, and animal virus systems. We have tested several transcriptional regulatory sequences for affecting DNA replication, based on pUC vector, in transient systems. Autonomous replication of transfected plasmids was assayed by PCR amplification of the fragments derived from the plasmids, which had replicated in mammalian cells. By this highly efficient method of detecting replicated molecules, pUC19, but not pUC18, showed a week replication activity in transfected cells. Nucleotide sequences around the HindIII site in pUC19 were required for replication. Monomers or dimers of the octamer transcription motif of the mouse immunoglobulin heavy chain gene, inserted in multicloning sites of pUC19, could stimulate replication, while the 4- or 6-mers did not, in contrast to the results on its transcription activity. Other transcriptional elements including AP1, HSE, and E2F also stimulated replication, but neither CRE nor Sp1 binding motif did. These results suggest that at least some of the transcriptional regulatory sequences function as-modulators of DNA replication as well as of transcription.

A novel DNA replication origin identified in the human heat shock protein 70 gene promoter.

A general and sensitive method for the mapping of initiation sites of DNA replication in vivo, developed by Vassilev and Johnson, has revealed replication origins in the region of simian virus 40 ori, in the regions upstream from the human c-myc gene and downstream from the Chinese hamster dihydrofolate reductase gene, and in the enhancer region of the mouse immunoglobulin heavy-chain gene. Here we report that the region containing the promoter of the human heat shock protein 70 (hsp70) gene was identified as a DNA replication origin in HeLa cells by this method. Several segments of the region were cloned into pUC19 and examined for autonomously replicating sequence (ARS) activity. The plasmids carrying the segments replicated episomally and semiconservatively when transfected into HeLa cells. The segments of ARS activity contained the sequences previously identified as binding sequences for a c-myc protein complex (T. Taira, Y. Negishi, F. Kihara, S. M. M. Iguchi-Ariga, and H. Ariga, Biochem. Biophys. Acta 1130:166-174, 1992). Mutations introduced within the c-myc protein complex binding sequences abolished the ARS activity. Moreover, the ARS plasmids stably replicated at episomal state for a long time in established cell lines. The results suggest that the promoter region of the human hsp70 gene plays a role in DNA replication as well as in transcription.

A novel DNA replication origin identified in the human heat shock protein 70 gene promoter.

A general and sensitive method for the mapping of initiation sites of DNA replication in vivo, developed by Vassilev and Johnson, has revealed replication origins in the region of simian virus 40 ori, in the regions upstream from the human c-myc gene and downstream from the Chinese hamster dihydrofolate reductase gene, and in the enhancer region of the mouse immunoglobulin heavy-chain gene. Here we report that the region containing the promoter of the human heat shock protein 70 (hsp70) gene was identified as a DNA replication origin in HeLa cells by this method. Several segments of the region were cloned into pUC19 and examined for autonomously replicating sequence (ARS) activity. The plasmids carrying the segments replicated episomally and semiconservatively when transfected into HeLa cells. The segments of ARS activity contained the sequences previously identified as binding sequences for a c-myc protein complex (T. Taira, Y. Negishi, F. Kihara, S. M. M. Iguchi-Ariga, and H. Ariga, Biochem. Biophys. Acta 1130:166-174, 1992). Mutations introduced within the c-myc protein complex binding sequences abolished the ARS activity. Moreover, the ARS plasmids stably replicated at episomal state for a long time in established cell lines. The results suggest that the promoter region of the human hsp70 gene plays a role in DNA replication as well as in transcription.

Regulation of adenovirus and cellular gene expression and of cellular transformation by the E1B-encoded 175-amino-acid protein.

Mutants of type 5 adenovirus that fail to express the E1B-gene-encoded 175-amino-acid (175R) protein are unable to morphologically transform primary or continuous cultures of rat embryo fibroblast cells. This phenotype could result from a direct effect of this E1B polypeptide (along with E1A polypeptides) on cellular gene expression resulting in a pathway leading to altered cell growth or from an indirect role of the 175R protein made possible by its ability to modulate viral early-gene (most likely E1A) expression. To distinguish between these two models, viruses were constructed that expressed the individual E1A 13S and 12S genes in the presence of either the E1B 175R or 495R protein. Regardless of the E1A gene product that was expressed, viruses that failed to express the E1B 175R protein were transformation defective. Additional studies suggest that the E1A 289R protein and E1B 495R protein function in a common pathway leading to the establishment of the transformed cell. We also observe that E3 gene expression by viruses that fail to express the E1A 289R protein affects the efficiency of focus formation. When tested in both nonpermissive CREF cells and permissive HeLa cells, the lack of 175R protein expression appeared to have no effect (a transient twofold decrease in E1A mRNA accumulation was observed in CREF cells) on viral early-gene expression. These results suggest that the initiation of the transformed cell phenotype occurs because of some interaction in a common pathway between the viral E1A proteins and E1B 175R protein. Furthermore, we have shown that the E1B 175R protein does not enhance the rate of transcription initiation from the mouse immunoglobulin heavy chain gene promoter when these sequences are localized on a viral genome, and it does not diminish the ability of the E1A proteins to decrease the rate of enhancer-dependent transcription.

DNA bending induced by specific interaction of decamer binding proteins with immunoglobulin gene control sequences

In order to investigate the properties of specific DNA-binding proteins involved in tissue-specific regulation of immunoglobulin genes, we have analyzed the interaction of nuclear proteins from mouse B-cell hybridomas with promoter and enhancer sequences of a mouse immunoglobulin heavy chain gene. Visualization of specific complexes has shown that protein binding induces a sharp bend at the position of the conserved decamer sequence. After fractionation of nuclear extracts, several sequence-specific DNA binding proteins could be distinguished by UV crosslinking to radioactive synthetic oligonucleotides. Decamer binding factor I (DBF-I), a protein of 100–105 kDa, and DBF-II, a family of proteins of 25–30 kDa, were purified on specific DNA-affinity columns. Both proteins bend the DNA at the dc sequence, as shown by electron microscopy and by gel retardation. These data suggest that one possible function of sequence-specific regulatory proteins may be to locally change the DNA topology, thereby facilitating the interaction of additional transcription factors with the primary complex.

Recombination within the upstream gene of duplicated myelin basic protein genes of myelin deficient shimld mouse results in the production of antisense RNA.

The myelin deficient shimld mouse is an autosomal recessive mutant, characterized by hypomyelination in the central nervous system. The expression of the myelin basic protein (MBP) gene is inhibited transcriptionally. The MBP gene is duplicated tandemly in mld, and exons 3 to 7 of the upstream copy is inverted. In the present studies, we determined the approximate position of the 5' boundary and the nucleotide sequence surrounding the 3' boundary of the inversion and found a number of sequences homologous to the switching regions of mouse immunoglobulin heavy chain gene and J regions of human T cell receptor genes. Antisense RNA complementary to exons 3 and 7, which correspond to the inverted segment, was detected by RNase protection studies. This abnormal transcript was also shown to elongate through the inverted segment to reach the transcription initiation site of the downstream gene.

A lymphoproliferative abnormality associated with inappropriate expression of the Thy-1 antigen in transgenic mice

The Thy-1 antigen is a cell-surface glycoprotein of unknown function expressed on mouse T lymphocytes, neurons, and hematopoietic stem cells. To alter the normal pattern of Thy-1 expression during hematopoietic differentiation, we created transgenic mice using a hybrid Thy-1 gene containing a transcriptional enhancer of the mouse immunoglobulin heavy chain gene (Eμ). Strains of mice bearing the Thy-1.2/Eμ gene express the Thy-1.2 antigen on mature B lymphocytes and their progenitors, and develop a heritable lymphoid hyperplasia characterized by massive expression of the Thy-1.2 antigen in the bone marrow and lymph nodes. The phenotype associated with inappropriate developmental regulation of the Thy-1 gene suggests that the Thy-1 antigen may play a role inducing activation or differentiation events on early lymphocyte progenitor cells.

The mouse immunoglobulin heavy-chain gene enhancer contains sequences that inhibit transcription in vitro in HeLa cell extracts.

The effect of the cell-specific mouse immunoglobulin heavy-chain gene (IgH) enhancer on transcription from heterologous promoter elements was studied in vitro with a HeLa whole-cell extract. No stimulation of transcription could be seen under conditions in which an activation was observed with the simian virus 40 enhancer. We found, however, that a specific segment of the IgH enhancer region contains sequences which inhibit transcription in vitro.

The mouse immunoglobulin heavy-chain gene enhancer contains sequences that inhibit transcription in vitro in HeLa cell extracts.

The effect of the cell-specific mouse immunoglobulin heavy-chain gene (IgH) enhancer on transcription from heterologous promoter elements was studied in vitro with a HeLa whole-cell extract. No stimulation of transcription could be seen under conditions in which an activation was observed with the simian virus 40 enhancer. We found, however, that a specific segment of the IgH enhancer region contains sequences which inhibit transcription in vitro.

The mouse immunoglobulin heavy-chain enhancer: effect on transcription in vitro and binding of proteins present in HeLa and lymphoid B cell extracts.

The ability of the mouse immunoglobulin heavy chain gene (IgH) enhancer to stimulate in vitro transcription from the adenovirus-2 major late promoter (Ad2MLP) has been investigated. In agreement with the in vivo cell-type specificity of this enhancer, a stimulation can be observed in BJA-B lymphoid B cell, but not in HeLa cell, extracts. Under identical conditions, the Simian virus 40 (SV40) enhancer stimulates transcription in both extracts to approximately the same extent. In addition we have found that a sequence, previously shown to inhibit transcription in vitro in HeLa cell extracts, is also inhibitory in B cell extracts. DNase I footprint and DMS-methylation protection experiments indicate that each cell type contains proteins which bind to specific sequences of the IgH enhancer. The relationship between the binding of these proteins and the preferential activity of the IgH enhancer in B cells is discussed.

Specific interaction of cellular factors with the B enhancer of polyoma virus.

Specific interactions between proteins from mouse 3T6 cells and the enhancer sequence of polyoma virus were detected using the method of band shifting on polyacrylamide gels. Proteins eluted from 3T6 nuclei using a buffer containing 0.55 M NaCl, formed a stable complex with the B enhancer of polyoma virus. At least two different factors are involved in this interaction. The contact sites which were mapped on the DNA sequence using DNase I footprinting correspond to a GC-rich palindrome surrounded by two sequences homologous respectively to the immunoglobulin and to the immunoglobulin and SV40 enhancers. Moreover Bal31 deletion analysis confirmed that similar sequences are required for the formation of the complex. In spite of a common function and partial sequence homology among some enhancers, neither the polyoma A enhancer, the mouse immunoglobulin heavy chain gene enhancer, nor the origin-promoter-enhancer region of SV40 efficiently competed with the polyoma B enhancer for the binding of these molecules.

Accurate and efficient transcription of human c-myc genes injected into Xenopus laevis oocytes.

We investigated the expression of the cloned human c-myc gene in Xenopus laevis oocytes microinjected with different recombinants. We found that microinjected plasmid DNA carrying an intact human c-myc gene directs efficient and faithful transcription from its own two promoters in X. laevis oocytes. This active transcription was unaffected by the presence of previously identified enhancing elements such as simian virus 72-base pair repeats or mouse immunoglobulin heavy-chain gene enhancer sequences in the construct in cis. This suggests that all necessary DNA sequences for accurate and faithful transcription recognized by the transcription machinery of the frog oocyte are self-contained. In addition, we have found that human c-myc transcripts synthesized in oocytes are properly polyadenylated at either one of two sites and also that the transcripts are spliced correctly but with low efficiency.

Accurate and efficient transcription of human c-myc genes injected into Xenopus laevis oocytes.

We investigated the expression of the cloned human c-myc gene in Xenopus laevis oocytes microinjected with different recombinants. We found that microinjected plasmid DNA carrying an intact human c-myc gene directs efficient and faithful transcription from its own two promoters in X. laevis oocytes. This active transcription was unaffected by the presence of previously identified enhancing elements such as simian virus 72-base pair repeats or mouse immunoglobulin heavy-chain gene enhancer sequences in the construct in cis. This suggests that all necessary DNA sequences for accurate and faithful transcription recognized by the transcription machinery of the frog oocyte are self-contained. In addition, we have found that human c-myc transcripts synthesized in oocytes are properly polyadenylated at either one of two sites and also that the transcripts are spliced correctly but with low efficiency.

Genomic sequencing.

Unique DNA sequences can be determined directly from mouse genomic DNA. A denaturing gel separates by size mixtures of unlabeled DNA fragments from complete restriction and partial chemical cleavages of the entire genome. These lanes of DNA are transferred and UV-crosslinked to nylon membranes. Hybridization with a short 32P-labeled single-stranded probe produces the image of a DNA sequence "ladder" extending from the 3' or 5' end of one restriction site in the genome. Numerous different sequences can be obtained from a single membrane by reprobing. Each band in these sequences represents 3 fg of DNA complementary to the probe. Sequence data from mouse immunoglobulin heavy chain genes from several cell types are presented. The genomic sequencing procedures are applicable to the analysis of genetic polymorphisms, DNA methylation at deoxycytidines, and nucleic acid-protein interactions at single nucleotide resolution.

A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes

Transcriptional enhancers, originally discovered in viral genomes, are short, cis-acting, regulatory sequences that strongly stimulate transcription from promoters of nearby genes. We demonstrate the existence of an enhancer within a mouse immunoglobulin heavy chain gene. A DNA fragment located between the joining region and the switch recombination region in the intron upstream of the immunoglobulin μ constant region has been linked, in both orientations, to genes coding for rabbit β-globin or SV40 T antigen. This element enhances the number of correct β-globin gene transcripts by at least two orders of magnitude and also stimulates production of T antigen. It acts from several hundred to several thousand base pairs up or downstream of a promoter without amplifying template copy number. Of the various cell lines tested, the immunoglobulin gene enhancer functions only in lymphocyte-derived (myeloma) cells. We propose that this tissue-specific enhancer contributes to the activation of somatically rearranged immunoglobulin variable region genes and possibly to abnormal expression of other genes (e.g. c- myc) that become translocated to its domain of influence.

Diversity and joining segments of mouse immunoglobulin heavy chain genes are closely linked and in the same orientation: implications for the joining mechanism.

We have mapped 12 diversity (D) gene segments of mouse Ig heavy chains between 80 and 1 kilobases 5' to the four joining (JH) gene segments by molecular analysis of DNA clones isolated from recombinant phage and cosmid libraries. All identified D and JH segments are in the same 5'-3' orientation. The significance of these findings with respect to the joining mechanism for Ig gene segments is discussed.

Organization, structure, and assembly of immunoglobulin heavy chain diversity DNA segments.

We have identified, cloned, and sequenced eight different DNA segments encoding the diversity (D) regions of mouse immunoglobulin heavy-chain genes. Like the two D segments previously characterized (16, 17), all eight D segments are flanked by characteristic heptamers and nonamers separated by 12-bp spacers. These 10 D segments, and several more D segments identified but not yet sequenced, can be classified into three families based on the extent of sequence homology. The SP2 family consists of nine highly homologous D segments that are all 17-bp long and clustered in a chromosomal region of approximately 60 kb. The FL16 family consists of up to four D segments, two of which were mapped in the 5' end region of the SP2-D cluster. The two FL16D segments are 23 and 17 bp long. The third, the Q52 family, is a single-member family of the 10-bp-long DQ52, located 700 bp 5' to the JH cluster. We argue that the D-region sequences of the majority of heavy chain genes arise from these germline D segments by various somatic mechanisms, including joining of multiple D segments. We present a specific model of D-D joining that does not violate the 12/23-bp spacer rule.