Copyright (c) 2010 All rights reserved. http://works.bepress.com/stavnezerj Recent documents in Janet M. Stavnezer en-us Fri, 29 Jan 2010 03:27:54 PST 3600 http://works.bepress.com/stavnezerj/91 http://works.bepress.com/stavnezerj/91 Wed, 27 Jan 2010 12:05:29 PST Antibody class switching occurs in mature B cells in response to antigen stimulation and costimulatory signals. It occurs by a unique type of intrachromosomal deletional recombination within special G-rich tandem repeated DNA sequences [called switch, or S, regions located upstream of each of the heavy chain constant (C(H)) region genes, except Cdelta]. The recombination is initiated by the B cell-specific activation-induced cytidine deaminase (AID), which deaminates cytosines in both the donor and acceptor S regions. AID activity converts several dC bases to dU bases in each S region, and the dU bases are then excised by the uracil DNA glycosylase UNG; the resulting abasic sites are nicked by apurinic/apyrimidinic endonuclease (APE). AID attacks both strands of transcriptionally active S regions, but how transcription promotes AID targeting is not entirely clear. Mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-strand breaks with DNA ends appropriate for end-joining recombination. Proteins required for the subsequent S-S recombination include DNA-PK, ATM, Mre11-Rad50-Nbs1, gammaH2AX, 53BP1, Mdc1, and XRCC4-ligase IV. These proteins are important for faithful joining of S regions, and in their absence aberrant recombination and chromosomal translocations involving S regions occur. Janet Stavnezer Animals Cytidine Deaminase Germinal Center Humans Immunoglobulin Class Switching Immunoglobulin Isotypes Models, Genetic *Recombination, Genetic http://works.bepress.com/stavnezerj/90 http://works.bepress.com/stavnezerj/90 Wed, 27 Jan 2010 12:05:27 PST Antibody class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded breaks (DSBs) in switch-region DNA. The initial steps in DSB formation have been elucidated, involving cytosine deamination by activation-induced cytidine deaminase and generation of abasic sites by uracil DNA glycosylase. However, it is not known how abasic sites are converted into single-stranded breaks and, subsequently, DSBs. Apurinic/apyrimidinic endonuclease (APE) efficiently nicks DNA at abasic sites, but it is unknown whether APE participates in CSR. We address the roles of the two major mammalian APEs, APE1 and APE2, in CSR. APE1 deficiency causes embryonic lethality in mice; we therefore examined CSR and DSBs in mice deficient in APE2 and haploinsufficient for APE1. We show that both APE1 and APE2 function in CSR, resulting in the DSBs necessary for CSR and thereby describing a novel in vivo function for APE2. Jeroen E. J. Guikema Animals B-Lymphocyte Subsets *DNA Damage DNA-(Apurinic or Apyrimidinic Site) Lyase Exodeoxyribonucleases Gene Expression Regulation Immunoglobulin Class Switching Lymphocyte Activation Mice Mice, Knockout Recombination, Genetic Spleen http://works.bepress.com/stavnezerj/89 http://works.bepress.com/stavnezerj/89 Tue, 01 Apr 2008 10:37:17 PDT TGF-beta1 directs class switching to IgA by splenic B cells and by the surface IgM+ B cell line, I.29mu, by inducing germline (GL) Ig alpha transcripts. The promoter segment between -130 and +46, relative to the first initiation site for mouse GL alpha transcripts, is sufficient for expression and TGF-beta1 inducibility of a reporter gene in B cell lines. Within this segment resides a TGF-beta1-responsive element (TbetaRE) that is required for induction of the promoter by TGF-beta1 and, when multimerized, is sufficient to transfer TGF-beta1 inducibility to another promoter. In this report we show that a TGF-beta1-inducible complex binds the TbetaRE and contains the transcription factor core-binding factor (CBF; also known as acute myeloid leukemia, AML). Although all three CBF alpha family members activate the GL alpha promoter, only CBF alpha3 (AML-2) is induced by TGF-beta1 in splenic B and I.29mu cells. The TbetaRE contains two CBF binding sites. Mutation of both sites reduces but does not eliminate induction of the GL alpha promoter by TGF-beta1 or by overexpression of CBF, possibly due to the presence of an additional CBF site in the promoter. In addition, the TbetaRE contains two copies of another sequence motif. Mutation of these motifs eliminates TGF-beta1 induction of the GL alpha promoter. Together the data indicate that TGF-beta1 induction of the alpha promoter involves induction of CBF alpha3, which binds to the TbetaRE of the promoter along with one or more proteins. M. J. Shi Animals B-Lymphocytes Core Binding Factors DNA-Binding Proteins Gene Expression Regulation *Genes, Immunoglobulin Genes, Reporter Immunoglobulin A Immunoglobulin Class Switching Immunoglobulin M Lymphoma, B-Cell Macromolecular Substances Mice *Neoplasm Proteins *Promoter Regions (Genetics) Receptors, Antigen, B-Cell Spleen Transcription Factors Transforming Growth Factor beta Tumor Cells, Cultured http://works.bepress.com/stavnezerj/88 http://works.bepress.com/stavnezerj/88 Tue, 01 Apr 2008 10:37:12 PDT A large body of data indicate that antibody class switching is directed by cytokines by inducing or repressing transcription from unrearranged, or germline, CH genes. Interleukin 4 (IL-4) induces transcription of the germline C epsilon genes in activated B cells and subsequently, cells in this population will undergo switch recombination to immunoglobulin E. Furthermore, the data suggest that transcription of germline C epsilon genes is required for class switching. In this paper we define DNA elements required for induction of transcription of the germline C epsilon genes by IL-4. To do this, segments of DNA from the 5' flank of the initiation sites for germline epsilon RNA were ligated to a luciferase reporter gene and transfected into two mouse B cell lines, one of which can be induced to switch to IgE. By analysis of a series of 5' deletion constructs and linker-scanning mutations, we demonstrate that a 46-bp segment (residing at -126/-79 relative to the first RNA initiation site) contains an IL-4 responsive region. By electrophoretic mobility shift assays, we find that this segment binds three transcription factors: the recently described NF-IL4, one or more members of the C/EBP family of transcription factors, and NF-kappa B/p50. Mutation of any of the binding sites for these three factors abolishes or reduces IL-4 inducibility of the epsilon promoter. A 27-bp segment within this IL-4 response region containing binding sites for NF-IL4 and a C/EBP factor is sufficient to transfer IL-4 inducibility to a minimal c-fos promoter. S. Delphin Animals Base Sequence Binding Sites CCAAT-Enhancer-Binding Proteins Consensus Sequence DNA-Binding Proteins *Genes, Immunoglobulin Humans Immunoglobulin epsilon-Chains Interleukin-4 Mice Molecular Sequence Data NF-kappa B Nuclear Proteins Oligodeoxyribonucleotides *Promoter Regions (Genetics) Sequence Alignment Sequence Homology, Amino Acid Structure-Activity Relationship Transcription Factors Transcription, Genetic http://works.bepress.com/stavnezerj/87 http://works.bepress.com/stavnezerj/87 Tue, 01 Apr 2008 10:37:07 PDT Antibody class switching is regulated by transcription of unrearranged C(H) genes to produce germline (GL) transcripts which direct the choice of isotype and are required for switching. However, their role is unknown. GL transcripts are initiated at the I exons located upstream of each switch region. Although deletion of the I exon by gene targeting prevents switch recombination to that CH gene, the Ialpha exon can be replaced by an entirely different DNA segment, a minigene driven by the phosphoglycerate kinase (PGK) promoter and encoding hypoxanthine phosphoribosyl transferase (HPRT), oriented in the sense direction, without reducing antibody class switching to IgA. To understand why HPRT substitution of the Ialpha exon does not disrupt switch recombination, we have analyzed the structure of the transcript from the targeted allele in these mice. We identify a spliced transcript in which the HPRT exons are spliced to the C(alpha) gene segments, resulting in a structure similar to normal GL transcripts. The abundance of this transcript is similar to that of the normal alpha GL RNA. We also demonstrate that switching to the four IgG subclasses in B cells from these mice is reduced in comparison to wild-type mice. We discuss the possibility that the strong PGK promoter inserted at the Ig alpha locus may interfere with interaction of the promoters for gamma GL transcripts with the 3' IgH enhancer. G. Qiu Animals Exons Gene Expression Regulation Germ Cells Hypoxanthine Phosphoribosyltransferase Immunoglobulin A *Immunoglobulin Class Switching Immunoglobulin G Immunoglobulin Heavy Chains Mice Mice, Knockout *RNA Splicing Recombination, Genetic Sequence Analysis, DNA http://works.bepress.com/stavnezerj/86 http://works.bepress.com/stavnezerj/86 Tue, 01 Apr 2008 10:37:02 PDT Ig heavy chain class switch recombination (CSR) determines the expression of Ig isotypes. The molecular mechanism of CSR and the factors regulating this process have remained elusive. Recombination occurs primarily within switch (S) regions, located upstream of each heavy chain gene (except Cdelta). These repetitive sequences contain consensus DNA-binding sites for the DNA-binding protein late SV40 factor (LSF) (CP2/leader-binding protein-1c). In this study, we demonstrate by EMSA that purified rLSF, as well as LSF within B cell extracts, directly binds both Smu and Salpha sequences. To determine whether LSF is involved in regulating CSR, two different LSF dominant negative variants were stably expressed in the mouse B cell line I.29 mu, which can be induced to switch from IgM to IgA. Overexpression of these dominant negative LSF proteins results in decreased levels of endogenous LSF DNA-binding activity and an increase in cells undergoing CSR. Thus, LSF represses class switching to IgA. In agreement, LSF DNA-binding activity was found to decrease in whole cell extracts from splenic B cells induced to undergo class switching. To elucidate the mechanism of CSR regulation by LSF, the interactions of LSF with proteins involved in chromatin modification were tested in vitro. LSF interacts with both histone deacetylases and the corepressor Sin3A. We propose that LSF represses CSR by histone deacetylation of chromatin within S regions, thereby limiting accessibility to the switch recombination machinery. Elise E. Drouin Amino Acid Substitution Animals Base Sequence *Binding Sites, Antibody Cell Line Chromatin DNA-Binding Proteins Gene Silencing Glutamic Acid Glutamine Humans Immunoglobulin A *Immunoglobulin Class Switching *Immunoglobulin Switch Region Immunoglobulin alpha-Chains Immunoglobulin mu-Chains Leucine Lymphoma, B-Cell Lysine Mice Mice, Inbred C57BL Molecular Sequence Data RNA-Binding Proteins Repressor Proteins Spleen Transcription Factors Transcription, Genetic Tumor Cells, Cultured http://works.bepress.com/stavnezerj/85 http://works.bepress.com/stavnezerj/85 Tue, 01 Apr 2008 10:36:57 PDT No abstract provided. W. Dunnick Animals B-Lymphocytes Gene Expression Regulation *Immunoglobulin Switch Region Mice Mice, Transgenic Recombination, Genetic Transcription, Genetic http://works.bepress.com/stavnezerj/84 http://works.bepress.com/stavnezerj/84 Tue, 01 Apr 2008 10:36:53 PDT No abstract provided. Janet Stavnezer Animals B-Lymphocytes Gene Expression Regulation *Genes, Regulator *Genes, Switch Immunoglobulin A Immunoglobulin E Immunoglobulin G Immunoglobulin Heavy Chains Mice Transcription, Genetic Tumor Cells, Cultured http://works.bepress.com/stavnezerj/83 http://works.bepress.com/stavnezerj/83 Tue, 01 Apr 2008 10:36:49 PDT No abstract provided. E. Severinson Animals B-Lymphocytes Cells, Cultured Enzyme-Linked Immunosorbent Assay Humans Immunoglobulin E Interleukin-4 Interleukin-5 Lipopolysaccharides Mice RNA, Messenger T-Lymphocytes http://works.bepress.com/stavnezerj/82 http://works.bepress.com/stavnezerj/82 Tue, 01 Apr 2008 10:36:46 PDT Mismatch repair proteins participate in antibody class switch recombination, although their roles are unknown. Previous nucleotide sequence analyses of switch recombination junctions indicated that the roles of Msh2 and the MutL homologues, Mlh1 and Pms2, differ. We now asked if Msh2 and Mlh1 function in the same pathway during switch recombination. Splenic B cells from mice deficient in both these proteins were induced to undergo switching in culture. The frequency of switching is reduced, similarly to that of B cells singly deficient in Msh2 or Mlh1. However, the nucleotide sequences of the Smu-Sgamma3 junctions resemble junctions from Mlh1- but not from Msh2-deficient cells, suggesting Mlh1 functions either independently of or before Msh2. The substitution mutations within S regions that are known to accompany switch recombination are increased in Msh2- and Mlh1 single-deficient cells and further increased in the double-deficient cells, again suggesting these proteins function independently in class switch recombination. The finding that MMR functions to reduce mutations in switch regions is unexpected since MMR proteins have been shown to contribute to somatic hypermutation of antibody variable region genes. Carol E. Schrader Adenosine Triphosphatases Animals Base Pair Mismatch Base Sequence Carrier Proteins DNA Repair *DNA Repair Enzymes DNA-Binding Proteins Immunoglobulin Class Switching Mice Molecular Sequence Data MutS Homolog 2 Protein Neoplasm Proteins Nuclear Proteins Proto-Oncogene Proteins *Recombination, Genetic http://works.bepress.com/stavnezerj/81 http://works.bepress.com/stavnezerj/81 Tue, 01 Apr 2008 10:36:40 PDT B cell-specific activator protein (BSAP)/Pax-5 is a paired domain DNA-binding protein expressed in the developing nervous system, testis, and in all B lineage cells, except terminally differentiated plasma cells. BSAP regulates transcription of several genes expressed in B cells and also the activity of the 3' IgH enhancer. As it has binding sites within or 5' to the switch regions of nearly all Ig heavy chain C region genes and also is known to increase transcription of the germline epsilon RNA, BSAP has been hypothesized to be involved in regulation of Ab class switch recombination. To directly examine the effects of BSAP on isotype switching, we use a tetracycline-regulated expression system to overexpress BSAP in the surface IgM+ I.29 mu B cell line, a mouse cell line that can be induced to undergo class switch recombination. We find that overexpression of BSAP inhibits switching to IgA in I.29 mu cells stimulated with LPS + TGF-beta 1 + nicotinamide, but enhances switching to IgE in cells stimulated with LPS + IL-4 + nicotinamide. Parallel to its effects on switching, overexpression of BSAP inhibits germline alpha RNA expression and the transcriptional activity of the germline alpha promoter, while enhancing activity of the germline epsilon promoter. Proliferation of I.29 mu cells is not affected in this system. The possible mechanisms and significance of the effect of BSAP on isotype switching are discussed. G. Qiu Adjuvants, Immunologic Animals B-Cell-Specific Activator Protein B-Lymphocytes Base Sequence Binding Sites DNA-Binding Proteins Gene Expression Regulation Immunoglobulin A Immunoglobulin Class Switching Immunoglobulin E Immunoglobulin alpha-Chains Immunoglobulin epsilon-Chains Immunoglobulin mu-Chains Immunosuppressive Agents Lymphoma, B-Cell Mice Molecular Sequence Data Nuclear Proteins Promoter Regions (Genetics) Tetracycline *Transcription Factors Transfection Tumor Cells, Cultured http://works.bepress.com/stavnezerj/80 http://works.bepress.com/stavnezerj/80 Tue, 01 Apr 2008 10:36:35 PDT Signal transducer and activator of transcription 6 (Stat6) and NF-kappaB are widely distributed transcription factors which are induced by different stimuli and bind to distinct DNA sequence motifs. Interleukin-4 (IL-4), which activates Stat6, synergizes with activators of NF-kappaB to induce IL-4-responsive genes, but the molecular mechanism of this synergy is poorly understood. Using glutathione S-transferase pulldown assays and coimmunoprecipitation techniques, we find that NF-kappaB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. An IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4 when Stat6 and NF-kappaB proteins are coexpressed in human embryonic kidney 293 (HEK 293) cells. The same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-kappaB proteins in IL-4-stimulated I.29mu B lymphoma cells. Furthermore, Stat6 and NF-kappaB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-kappaB. We conclude that the direct interaction between Stat6 and NF-kappaB may provide a basis for synergistic activation of transcription by IL-4 and activators of NF-kappaB. Ching-Hung Shen Binding Sites Cell Line DNA Drug Synergism Glutathione Transferase Humans Interleukin-4 NF-kappa B NF-kappa B p50 Subunit Protein Binding Recombinant Fusion Proteins STAT6 Transcription Factor Signal Transduction Trans-Activators Transcription Factor RelA *Transcription, Genetic Tumor Cells, Cultured http://works.bepress.com/stavnezerj/79 http://works.bepress.com/stavnezerj/79 Tue, 01 Apr 2008 10:36:31 PDT Three different forms of alpha-chains are synthesized by BF0.3 and 615.2, two cloned cell lines derived from the murine B lymphoma 1.29. The three forms of alpha-chains differ in size, pI, cellular location, and rate of turnover. They were identified by means of lactoperoxidase-catalyzed radioiodination, internal 14C or 35S labeling, and immunofluorescence techniques as membrane-bound(alpha m), secreted (alpha s), and intracellular (alpha ic) proteins. Comparison of immunoglobulin products of the two lymphoma lines with those of a hybridoma cell line, Id 150, which secretes IgA of the 1.29 idiotype but lacks membrane IgA, confirmed the assignments of alpha m, alpha s, and alpha ic. Results of biosynthetic labeling of BF0.3, 615.2, and Id 150 in the presence and absence of tunicamycin suggest that the difference in m.w. and charge observed between alpha m and alpha s can be attributed to differences in primary amino acid structure rather than different degrees of glycosylation. R. Sitia Animals Antibody-Producing Cells B-Lymphocytes Cell Line Chemistry Electrophoresis, Polyacrylamide Gel Goats Immunoglobulin A *Immunoglobulin Heavy Chains *Immunoglobulin alpha-Chains Lymphoma Mice Mice, Inbred Strains Receptors, Antigen, B-Cell Tunicamycin http://works.bepress.com/stavnezerj/78 http://works.bepress.com/stavnezerj/78 Tue, 01 Apr 2008 10:36:26 PDT The molecular basis of a human immunoglobulin deficiency characterized by the complete absence of kappa chains has been investigated by nucleotide sequence analyses of a patient's kappa constant region (C kappa) genes. Both of his C kappa genes had a single point mutation, resulting in the loss of the invariant tryptophan from one allele and of an invariant cysteine from the other allele. These results indicate that neither of the patient's C kappa alleles encoded a kappa chain that could form a stable intradomain disulfide bond, although peculiarities in the expression of kappa chains in the patient's family suggest that other factors may be involved. Janet Stavnezer Animals Base Sequence DNA, Recombinant Genetic Engineering Humans Immunoglobulin kappa-Chains Immunologic Deficiency Syndromes Pedigree Rabbits http://works.bepress.com/stavnezerj/77 http://works.bepress.com/stavnezerj/77 Tue, 01 Apr 2008 10:36:22 PDT We have analyzed the structure of Ig kappa chain genes in B cell lines derived from a human individual who cannot synthesize any kappa chains, and whose Igs all contain lambda chains (1). We have characterized secondary DNA recombination events at two kappa alleles which have undergone misaligned V-J recombinations. One such secondary recombination has joined the flanking sequences of a V kappa and a J kappa 2 gene segment as if it were the reciprocal product of a V-J kappa 2 recombination, and resulted in the displacement of the recombined VJ kappa 1 gene segments from the C kappa locus. The non-rearranged form of the V kappa fragment which had recombined with the J kappa 2 flank was cloned. Nucleotide sequencing of this fragment identified a V kappa gene that differed by at least 38% from all previously sequenced human V kappa genes. The other V-J kappa segment analyzed has undergone a secondary recombination at a different site from that described above, at a site within the intervening sequence between the J kappa and C kappa gene segments, similar to the location of secondary recombinations which have occurred in lambda + B cell lines from mice and humans (2,3). These results prove that multiple recombinations can occur at one J kappa-C kappa locus. Janet Stavnezer B-Lymphocytes Base Sequence Cell Line Cloning, Molecular Dysgammaglobulinemia Humans Immunoglobulin J-Chains Immunoglobulin Light Chains Immunoglobulin Variable Region Immunoglobulin kappa-Chains *Recombination, Genetic http://works.bepress.com/stavnezerj/76 http://works.bepress.com/stavnezerj/76 Tue, 01 Apr 2008 10:36:18 PDT Immunoglobulin class switch recombination (SR) occurs by a B cell-specific, intrachromosomal deletional process between switch regions. We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities. The plasmids are novel in that they lack a eukaryotic origin of DNA replication. The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells. The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes. These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity. Finally, we provide evidence for two distinct switching activities which independently mediate mu-->alpha and mu-->gamma3 SR. A. Shanmugam Animals B-Lymphocytes Base Sequence Cell Line DNA DNA Primers Escherichia coli Immunoglobulin Isotypes *Immunoglobulin Switch Region Mice Mice, Inbred BALB C Mice, Nude Molecular Sequence Data Plasmids Polymerase Chain Reaction Transcription, Genetic Transfection Transformation, Genetic http://works.bepress.com/stavnezerj/75 http://works.bepress.com/stavnezerj/75 Tue, 01 Apr 2008 10:36:13 PDT Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cmu) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination. Uracil DNA glycosylase (UNG) is required for most CSR activity although its role is disputed. Here we use ligation-mediated PCR to detect DSBs in S regions in splenic B cells undergoing CSR. We find that the kinetics of DSB induction corresponds with AID expression, and that DSBs are AID- and UNG-dependent and occur preferentially at G:C basepairs in WRC/GYW AID hotspots. Our results indicate that AID attacks cytosines on both DNA strands, and staggered breaks are processed to blunt DSBs at the initiating ss break sites. We propose a model to explain the types of end-processing events observed. Carol E. Schrader Animals B-Lymphocytes Cytidine Deaminase DNA DNA Glycosylases Gene Expression Regulation Gene Rearrangement, B-Lymphocyte Immunoglobulin Constant Regions Immunoglobulin mu-Chains Mice Mice, Mutant Strains Recombination, Genetic Somatic Hypermutation, Immunoglobulin Spleen Uracil-DNA Glycosidase http://works.bepress.com/stavnezerj/74 http://works.bepress.com/stavnezerj/74 Tue, 01 Apr 2008 10:36:09 PDT Antibody class switch recombination (CSR) occurs after antigen activation of B cells. CSR is directed to specific heavy chain isotypes by cytokines and B cell activators that induce transcription from the unrearranged, or germline (GL), C(H) region genes. Transforming growth factor (TGF)-beta1 is essential for switch recombination to IgA due to its ability to induce transcription from GL Ig alpha genes. It has been shown that the promoters which regulate transcription of mouse and human GL alpha RNAs contain a TGF-beta1-responsive element that binds Smad and core binding factor (CBFalpha)/AML/PEBPalpha/RUNX: They also contain other elements which bind the transcription factors CREB, BSAP and Ets family proteins. In this manuscript we demonstrate that two tandem Ets sites in the mouse GL alpha promoter bind the transcription factors Elf-1 and PU.1, and that the 3' site is essential for expression of a luciferase reporter gene driven by the GL alpha promoter. Binding of Elf-1 to the GL alpha promoter is inducible by lipopolysaccharide in nuclear extracts from splenic B cells. An NF-kappaB site is identified, although it does not contribute to expression of the promoter in reporter gene assays. Since CSR to IgA is greatly reduced in NF-kappaB/p50-deficient mice, these data support the hypothesis that NF-kappaB has roles in switching in addition to regulation of GL transcription. Finally, we demonstrate that nocodazole, which disrupts microtubules that sequester Smad proteins in the cytoplasm, stimulates transcription from the GL alpha promoter. M. J. Shi Animals Antineoplastic Agents B-Lymphocytes Base Sequence Binding Sites Cells, Cultured DNA-Binding Proteins Gene Expression Regulation Germ-Line Mutation Hela Cells Humans Immunoglobulin A Lymphocyte Activation Mice Molecular Sequence Data NF-kappa B Nocodazole Nuclear Proteins Promoter Regions (Genetics) Proto-Oncogene Proteins Proto-Oncogene Proteins c-ets RNA Spleen Transcription Factors Transforming Growth Factor beta Tumor Cells, Cultured http://works.bepress.com/stavnezerj/73 http://works.bepress.com/stavnezerj/73 Tue, 01 Apr 2008 10:36:05 PDT Antibody class switching is mediated by a DNA recombination event that replaces the C mu gene with one of the other heavy (H) chain constant region (CH) genes located 3' to the C mu gene. The regulation of this process is essential to the immune response because different CH regions provide different biological functions. Correlative evidence indicates that the isotype (class) specificity of the switch is determined by the accessibility of specific CH genes as indicated by hypomethylation and transcriptional activity. For example, RNAs transcribed from specific unrearranged CH genes are induced prior to switching under conditions that promote subsequent switching to these same CH genes. The function of transcription of these germline CH genes is unknown. In this report, we describe the structure of RNA transcribed from unrearranged gamma 1 genes in mouse spleen cells treated with LPS plus a HeLa cell supernatant containing recombinant interleukin 4. The germline gamma 1 RNA is initiated at multiple start sites 5' to the tandem repeats of the gamma 1 switch (S gamma 1) region. As is true for analogous RNAs transcribed from unrearranged gamma 2b and alpha genes, the germline gamma 1 RNA has an I exon transcribed from the region 5' to S gamma 1 sequences, which is spliced at a unique site to the C gamma gene. The germline gamma 1 RNA has an open-reading frame (ORF) that potentially encodes a small protein 48 amino acid in length. M. Xu Amino Acid Sequence Animals Antibody Diversity Base Sequence DNA DNA, Recombinant *Genes, Immunoglobulin Genes, Switch Immunoglobulin gamma-Chains Interleukin-4 Mice Mice, Inbred BALB C Molecular Sequence Data Open Reading Frames RNA Spleen Transcription, Genetic http://works.bepress.com/stavnezerj/72 http://works.bepress.com/stavnezerj/72 Tue, 01 Apr 2008 10:36:00 PDT Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), which converts cytosines to uracils in switch (S) regions. Subsequent excision of dU by uracil DNA glycosylase (UNG) of the base excision repair (BER) pathway is required to obtain double-strand break (DSB) intermediates for CSR. Since UNG normally initiates faithful repair, it is unclear how the AID-instigated S region lesions are converted into DSBs rather than correctly repaired by BER. Normally, DNA polymerase beta (Polbeta) would replace the dC deaminated by AID, leading to correct repair of the single-strand break, thereby preventing CSR. We address the question of whether Polbeta might be specifically down-regulated during CSR or inhibited from accessing the AID-instigated lesions, or whether the numerous AID-initiated S region lesions might simply overwhelm the BER capacity. 2875We find that nuclear Polbeta levels are induced upon activation of splenic B cells to undergo CSR. When Polbeta(-/-) B cells are activated to switch in culture, they switch slightly better to IgG2a, IgG2b, and IgG3 and have more S region DSBs and mutations than wild-type controls. We conclude that Polbeta attempts to faithfully repair S region lesions but fails to repair them all. Xiaoming Wu Animals B-Lymphocytes/enzymology/*immunology Cytidine Deaminase/*metabolism Cytosine/metabolism DNA Polymerase beta/deficiency/genetics/*physiology DNA Primers Immunoglobulin A/immunology Immunoglobulin G/immunology Immunoglobulin Switch Region/*genetics/immunology Lipopolysaccharides/pharmacology Mice Mice, Inbred C57BL Mutation *Recombination, Genetic Reverse Transcriptase Polymerase Chain Reaction Uracil/metabolism Uracil-DNA Glycosidase/*metabolism