Acute viral infections in humans and mice induce T lymphocyte responses which mediate viral clearance and result in the establishment of immunological memory. The course of an immune response to acute viral infection is associated with an immune deficiency in the lymphocyte compartment. This is usually characterized by the inability of lymphocytes to productively respond to mitogen or recall antigen. This thesis examined the acute lymphocytic choriomeningitis virus (LCMV) infection of the mouse and showed that T lymphocytes isolated from acutely LCMV-infected mice underwent activation-induced apoptosis upon signalling through the T-cell receptor (TcR)-CD3 complex. Kinetic studies demonstrated that this sensitivity to apoptosis directly correlated with the induction of immune deficiency, as measured by impaired proliferation in response to anti-CD3 antibody or to concanavalin A. Cell cycling in interleukin-2 (IL-2) alone stimulated proliferation of LCMV-induced T cells without inducing apoptosis, but preculturing of T cells from acutely-infected mice in IL-2 accelerated apoptosis upon subsequent TcR-CD3 crosslinking. T lymphocytes isolated from mice after the acute infection were less responsive to IL-2, but IL-2 receptor-bearing T cells, presumably memory T cells, responding to IL-2 were primed in each case to die a rapid apoptotic death upon TcR-CD3 crosslinking. These results indicated that virus infection-induced unresponsiveness to T-cell mitogens is in part attributable to apoptosis of the activated lymphocytes and suggest that the sensitization of memory cells by IL-2 and other stimulatory cytokines induced during an acute infection will cause them to die upon antigen recognition, thereby impairing specific responses to nonviral (recall) antigens. The cytotoxic T lymphocyte (CTL) response to acute LCMV infection is characterized by a massive (10-20 fold) expansion of CD8+ cell number, which after clearance of virus declines in number and returns to levels present prior to infection. This thesis documents the presence of high levels of apoptotic lymphocytes in situ in the spleens of mice during the silencing of the immune response to acute LCMV infection. Apoptotic cells were detected by an in situ nucleotidyl transferase (ISNT) assay. Both T and B lymphocytes, as revealed by immunohistochemical analysis, are shown to be dying in vivo, the latter in clusters. A biphasic occurrence of apoptosis during the course of the acute infection was found, with an increase in numbers of apoptotic cells above background at day 3 post-infection, and at day 11 post-infection, a second more pronounced peak coincident with the decline of the CTL response to the infection and with the decrease in total spleen leukocyte number. Apoptosis in vivo was detected in lpr mice lacking Fas expression, a molecule involved in lymphocyte apoptosis. Fas expression thus may not be required for lymphocyte apoptosis in the context of an acute viral infection. Apoptosis in situ and the silencing of the CD8+ T lymphocyte response to acute LCMV infection were unaffected by the enforced lymphocyte-directed expression of Bcl-2, a protein blocking IL-2 deprivation-induced apoptosis of lymphocytes. Experiments aimed at addressing the role of Bcl-2-sensitive apoptotic pathways in the development of viral persistence revealed that high-dose infection of Bcl-2-transgenic mice results in death of the animals. Flow cytometric analysis showed an accumulation of Thy1.2+ T cells in the lungs of these animals, and the air spaces in the lungs were occluded with cellular and fluid infiltrates. These results suggest that the pathology seen in the Bcl-2-transgenic mice upon high-dose infection is perhaps immune response-mediated (an immunopathology). This is consistent with a role for Bcl-2-sensitive pathways of lymphocyte apoptosis in the pathogenesis of persistent LCMV infection. The in situ demonstration of apoptosis in spleens during infection provide direct in vivo evidence for the death of lymphocytes during the recovery from an acute viral infection. This indicates that apoptotic elimination of the population en masse is a mechanism for halting an antiviral immune response upon clearance of virus. Furthermore, the data argue that IL-2 deprivation-driven apoptosis, upon clearance of virus, of the expanded T lymphocyte compartment is not the major mechanism involved in the silencing of the T cell response to acute LCMV infection. Resolution of an acute immune response leads into the generation of longterm immunological memory. Since this thesis focussed on T cell responses in viral infection, it was important to characterize the in vivo state of memory CD8+ T cells. During acute LCMV infection, the majority of the LCMV-specific CTL activity tested immediately ex vivo was mediated by CD8+ L-selectin-Mac-1+ CTL. The L-selectin- population of CD8+ cells elicited during acute infection also carried >99% of the restimulatable CD8+ CTLp to LCMV, and these required added IL-2 for development into effectors in vitro. In contrast to the acute infection, most of the virus-specific CTLp in immune mice were L-selectin+. Examination of CD8+ T cells in LCMV-immune mice revealed that a L-selectin+ blast-sized population of cycling CD8+ cells contained CTLp which developed into effector CTL in the absence of added IL-2. These cells also expressed Mac-1 and IL-2R. Flow cytometric sorting for IL-2R+ and IL-2R-CD8+ cells in the immune animal revealed, by limiting dilution analysis, similar frequencies of CTLp in both populations. In bulk restimulation assays, the CD25+ CTLp did not require added IL-2 for their in vitro development into effectors, whereas the CD25- CTLp did. Hence, the different requirements for CTLp to effector development in vitro reflect qualitative differences in the in vivo state of the CTLp in the various subpopulations. LCMV-specific memory CTLp not requiring added IL-2 for differentiation were also found in the small-sized, non-cycling, CD8+L-selectin- cells. In contrast, the small-sized, non-cycling, CD8+L-selectin+, and CD8+IL-2R- populations also carried CTLp, but these required added IL-2 for development into effector CTL. Hence, T cell memory to LCMV is distributed among various lymphocyte subpopulations in immune animals, and the presence of an activated cycling cell component may account for the stability and long-term perpetuation of antiviral immunological memory. In summary, the susceptibility of activated T lymphocytes to apoptosis probably explains an aspect of virus-induced immune deficiency and allows for the establishment of homeostasis subsequent to the resolution of an acute viral infection.

The compaction of the eukaryotic genome into a complex, highly folded chromatin structure necessitates cellular mechanisms for allowing access of regulatory proteins to the DNA template. Recent advances have led to the identification of two distinct families of chromatin remodeling enzymes--multi-subunit complexes that harbor a SWI2/SNF2 ATPase family member, and the nuclear acetyltransferases. The Saccharomyces cerevisiae SWI/SNF complex, the prototype for the ATP-dependent chromatin remodeling machines, is required for expression of a subset of genes in yeast. This 2MDa multimeric assembly is believed to facilitate transcriptional enhancement by antagonizing chromatin-mediated transcriptional repression through disruption of histone-DNA contacts. In an attempt to identify components or regulators of the SWI/SNF complex, we have cloned three previously identified genes, ADA2, ADA3, and GCN5, that encode subunits of a complex distinct from SWI/SNF. During the course of this thesis work, one of these gene products, GCN5, was identified as the first catalytic nuclear histone acetyltransferase. The goal of this thesis work was to determine the role of the ADA/GCN5 complex in transcriptional activation in Saccharomyces cerevisiae. Using in vivo functional and genetic analysis, we have found that mutations in ADA2, ADA3, and GCN5 cause phenotypes strikingly similar to those of swi/snf mutants. ADA2, ADA3, and GCN5 are required for full expression of all SWI/SNF-dependent genes tested, including HO, SUC2, INO1, and Ty elements. Furthermore, mutations in the SIN1 gene, which encodes a non-histone chromatin component, or mutations in histones H3 or H4, alleviate the transcriptional defects caused by ada/gcn5 or swi/snf mutations. We have also found that ada2 swi1, ada3 swi1, and gcn5 swi1 double mutants are inviable and that mutations in SIN1 allow viability of these double mutants. To determine the biochemical activities of the native GCN5-containing complex in yeast, we have partially purified three chromatographically distinct GCN5-dependent acetyltransferase activities. We have found that these three acetyltransferase complexes demonstrate unique substrate specificities for free histones and histones assembled into nucleosomal arrays. Additionally, we found that these enzymes not only acetylate histones, but also purified yeast Sin1 protein, a non-histone chromatin component that resembles HMG1. We have also established a functional relationship between GCN5-dependent histone acetylation and polyamine-dependent chromatin condensation. We have found that depletion of cellular polyamines alleviates transcriptional defects caused by inactivation of the GCN5 histone acetyltransferase. In contrast, polyamine depletion does not alter the transcriptional requirements for the SWI/SNF chromatin remodeling complex. We have also found that polyamines facilitate oligomerization of nucleosomal arrays in vitro. Furthermore, this polyamine-mediated condensation reaction requires intact N-terminal domains of the core histones, and is inhibited by hyperacetylation of these domains. The results presented throughout this thesis support roles for the ADA/GCN5 products in antagonizing chromatin. In vivo analysis suggests a functional relationship between the ADA/GCN5 acetyltransferase complex (or complexes) and the SWI/SNF complex. These comp1exes may operate in concert at nucleosomes within specific promoters to facilitate activated transcription. Furthermore, our studies suggest that polyamines are repressors of transcription in vivo, and that an additional role of histone hyperacetylation is to antagonize the ability of polyamines to stabilize highly condensed states of chromosomal fibers.

The sequence of 1360 base pairs of the P22 PL operon was determined, linking a continuous sequence from PL through abc2. P22 mutants bearing deletions in the sequenced region were constructed and tested for their phenotypes. Plasmids were constructed to express PL operon genes singly and in combinations from PlacUV5. Two previously known genes, 17 and c3, are located within this sequence. In addition, three new genes have been identified: ral, kil and arf. Genes ral and c3 are homologous, as well as functionally analogous, to λ ral and cIII, respectively. P22 kil, like λ kil, kills the host cell when it is expressed. The two kil genes, although analogous in cell killing and map location, have no apparent sequence homology. The functions of the P22 and λ kil genes are unknown; however, P22 kil is essential for lytic growth in the absence of abc. Gene arf (accessory recombination function) is located just upstream of erf; it is essential for P22 growth in the absence of kil or other genes upstream in PL. The growth defect of P22 bearing a deletion that removes arf is complemented by expression of either arf or the λ red genes from plasmids. P22 sequences that include the stop codon for 17 potentially form a small stem-loop structure; these sequences are nearly identical to λ sequences that contain the stop codon for ssb. In λ this potential stem-loop structure occupies a map position near the terminator tL2b. Plasmids that include the potential P22 structure negatively regulate kil gene expression in cis.

The nucleolus is a plurifunctional organelle with dynamic protein exchange involved in diverse aspects of cell biology. Additionally, the nucleolus has been shown to have a role in the replication of numerous viruses, which includes HIV-1. Several groups have reported HIV-1 vRNA localization within the nucleolus. Moreover, it has been demonstrated the HIV-1 Rev protein localizes to the nucleolus and interacts with nucleolar proteins, including NPM1. Despite evidence for a nucleolar involvement during replication, a functional link has not been demonstrated. I investigated whether introncontaining vRNAs have a Rev-mediated nucleolar localization step prior to export. Furthermore, I examined whether NPM1 mediates Rev nucleolar localization, participates in Rev function, and/or post-transcriptional events during viral replication. I used coupled RNA fluorescence in situhybridization and indirect immunofluorescence to visualize intron-containing vRNA relative to the nucleolus in the absence or presence of Rev expression. An RNAi-based approach was used to examine the role of NPM1 in Rev function and viral replication in cell lines and primary human macrophages. My research findings support a model for a Rev-independent nucleolar localization step of introncontaining vRNA prior to export. Intriguingly, my results also suggest NPM1 does not participate in Rev nucleolar localization or Rev-mediated vRNA export, as previously proposed. Rather, my findings support a novel role for NPM1, the cytoplasmic localization and utilization of a select class of Rev-dependent vRNAs. Collectively, my findings provide novel insight for a functional role of the nucleolus and NPM1 in HIV-1 replication, which enhances our current understanding of HIV-1 biology.

The Insulin Receptor Substrate (IRS) proteins IRS-1 and IRS-2 are cytoplasmic adaptor proteins that organize and propagate intracellular signaling downstream of specific growth factor receptors, including the Insulin and Insulin-Like Growth Factor-1 Receptors (IR and IGF-1R, respectively). Despite sharing a high level of homology and the ability to stimulate Phosphotidylinositol-3-Kinase (PI3K) and Mitogen-Activated Protein Kinase (MAPK) signaling, IRS-1 and IRS-2 play distinct roles in mammary tumor progression. Specifically, IRS-1 promotes growth and proliferation, whereas IRS- 2 promotes motility, invasion, survival, aerobic glycolyis, and metastasis. To further understand the differences between IRS-1 and IRS-2, I investigated the mechanistic basis of IRS-2-mediated PI3K activation. I identified tyrosines in IRS-2 that mediate its recruitment and activation of PI3K in response to insulin and IGF-1 stimulation. Using a PI3K-binding deficient IRS-2 mutant, I demonstrated that IRS-2-dependent PI3K signaling promotes aerobic glycolysis through its ability to selectively regulate the phosphorylation of the Akt effector Glycogen Synthase Kinase-3β (Gsk-3β). I also performed a rigorous comparison of IRS-1 and IRS-2 signal transduction and their ability to regulate functions associated with tumor progression. These studies required the generation of a novel model system where IRS-1 and IRS-2 function could be compared in a genetically identical background. Using this model, I confirmed a role for IRS-1 in growth regulation and IRS-2 in tumor cell invasion, as well as expanded the understanding of differential IRS protein function by showing that IRS-2 more vi effectively promotes Akt activation. The model system I have established can be used for further characterization of IRS-1 and IRS-2-specific functions.

Cytoplasmic dynein has been implicated in a wide range of functions. Originally characterized as being responsible for retrograde axonal transport, its has also been shown to traffic vesicular organelles (Golgi, endosome and lysosome distribution), transport viral particles to the nucleus, and participate in microtubule organization. During mitosis, cytoplasmic dynein is thought to function in spindle pole focusing and prometaphase kinetochore capture. This thesis explores the mitotic roles of cytoplasmic dynein. The first chapter addresses the role of cytoplasmic dynein in kinetochore activity. Using immunofluoresence microscopy, a number of motors and related proteins were observed at the primary, but not secondary, constrictions of prometaphase multicentric chromosomes. The proteins assessed included the cytoplasmic dynein intermediate chains, three components of the dynactin complex (dynamitin, Arp1, and p150Glued), the kinesin related proteins CENP-E and MCAK, and the proposed structural and checkpoint proteins CENP-F, HZW10, and MAD2. The differential localization of these proteins offered new insight into the assembly and composition of both active and inactive centromeres, and provided a molecular basis for the apparent inactivity of the latter during chromosome segregation. The second chapter characterizes LIS1, a protein that is defective in the developmental brain disease type1 lissencephaly. Mutations in the LIS1 gene cause gross histological disorganization of the developing cerebral cortex resulting in a nearly smooth brain surface. Because genetic evidence from lower eukaryotes suggested that LIS1 acted within the cytoplasmic dynein pathway, it was of interest to analyze LIS1 in terms of cytoplasmic dynein function. LIS1 was found to coimmunoprecipitate with cytoplasmic dynein and its companion complex dynactin. During mitosis LIS1 localized to the prometaphase kinetochore, spindle microtubules and the cell cortex, known sites for cytoplasmic dynein binding. Interference with endogenous LIS1 in cultured mammalian cells displaced dynein localization and disrupted mitotic progression. LIS1 was concluded to participate in cytoplasmic dynein functions, but only during mitosis. Data presented in the final chapter further characterizes LIS1's interactions with microtubules, cytoplasmic dynein and the mammalian homologue of NUDC. LIS1 was not found to co-fractionate with microtubules, nor did overexpression of LIS1 cause visible effects on microtubule organization or dynamics. GFP-LIS1 was shown to ride along the plus ends of growing microtubules. Though LIS1 was not found to have a direct effect on microtubules, it may regulate dynein's microtubule binding activity. LIS1 was found to co-immunoprecipitate with a co-overexpressed cytoplasmic dynein subunit substantiating the existence of a dynein LIS1 supercomplex. Furthermore, association of these proteins increased markedly in mitotically blocked samples. LIS1's regulation of cytoplasmic dynein may change the capacity of the motor to efficiently manipulate its mitotic cargoes, dramatically effecting the timing of cell division. This disruption has implications for the fundamental role of cytoplasmic dynein during early embryonic development.

Cytokine induced transcription of the germline immunoglobulin heavy chain gene directs isotype switch recombination to that gene. Therefore, understanding the regulation of germline transcription is an important first step in understanding the class switching process. Treatment of human B cells with IL-4 results in germline epsilon transcription. Subsequent activation of a second signal is necessary for these cells to undergo class switch recombination and express surface IgE. In contrast, treatment of splenic murine B-cells with IL-4 alone does not induce germline epsilon transcription. However, treatment with IL-4 plus LPS does induces germline epsilon transcription, followed by class switching to the IgE isotype. In both human and mouse, IL-4 is absolutely required for induction of germline transcripts and expression of IgE. Therefore, IL-4 is considered to be an IgE switch factor. The murine B lymphoma line, I.29μ is an IgM+ B cell line which can be induced to switch to the IgE isotype by treatment with IL-4 plus LPS. In these cells, germline epsilon transcription is constitutive and can be further induced 5-20 fold with IL-4, whereas LPS has no effect at the RNA level. Thus, the I.29μ cell line provides a model system to study the regulatory effects of IL-4 on the murine germline epsilon promoter. The aim of this thesis is to characterize the murine germline epsilon promoter and identify the minimal DNA elements necessary and sufficient for IL-4 induction. To identify the promoter elements, two kb of the 5' flanking region to the first exon (Iε) of the germline epsilon transcript was cloned into a Luciferase reporter plasmid and assayed for promoter activity. Assay of successive 5' deletion mutations by transfections into two B cell lines, I.29μ and M12.4.1, identified the 213 bp promoter construct, -162Luc, as containing sufficient sequence to confer full promoter function. Assay of the linker scanning mutations in the -162Luc plasmid localized the IL-4 responsive effect to a 46 bp region of the promoter. This region contains three nuclear factor binding elements: a C/EBP site, a recently identified NF-IL-4 site and a NFкB/p50 site. In order to detect protein complexes that specifically interact with this active region of DNA, electrophoretic mobility shift assays were performed using double stranded, oligonucleotide probes of this IL-4 responsive region. An IL-4 inducible complex was identified in nuclear extracts of I.29μ as well as murine splenic B-cells. Competition experiments with mutant probes mapped this inducible complex to the NF-IL-4 site. Constitutive binding of both C/EBP and NFкB/p50 was demonstrated by cold competition and supershift experiments. Transfection experiments using a series of linker scanning mutations allowed identification of DNA elements necessary for IL-4 induction. In order to test if these elements are sufficent for IL-4 induction, double stranded oligonucleotides containing these elements were transfered to a minimal fos promoter plasmid and assayed for IL-4 responsiveness. A 27 bp fragment containing two DNA elements, a C/EBP and a NF-IL-4 site were sufficient to confer IL-4 inducibility to a minimal c-fos promoter. This study defined a different IL-4 response element in the murine germline epsilon promoter from that previously published. This IL-4 response element is identical to the IL-4 response element in the human germline epsilon promoter. The NF-IL-4 site is also present in the promoter of the IL-4 responsive gene, CD23b (FcεRII), and this element binds an IL-4 inducible complex present in the human monocytic cell line U937. Various reports demonstrate the presence of an IL-4 inducible complex by gel shift assays and indeed the binding activity of NF-IL-4 has been mapped to a 9 bp consensus sequence within a 19 bp fragment. However, the transfer of IL-4 inducibility has not been reported using fragments smaller than 123 bp, the importance of which is underscored by the fact that more than one factor is involved in this induction. The contribution of this thesis to the understanding of transcriptional induction by IL-4 is in the delineation of the factors involved - namely, a member of the C/EBP family and NF-IL-4 are required for IL-4 induction and NFкB/p5O modulates this induction.

Transcription initiation is a key step at which gene expression can be regulated. The sigma subunit of RNA polymerase provides the enzyme with the ability to recognize promoter sequences and initiate transcription at specific sites on the chromosome. The molecular basis of sigma function is not well known. It has been suggested that sigma factors confer promoter specificty by making direct contacts to the promoter DNA (Losick and Pero, 1981). To test this idea, suppressors of promoter down mutations were sought that affected the promoter recogniton properties of the σ70 subunit of E. coli RNA polymerase. Four such sigma mutants were obtained, two of which are allele-specific. One of these mutants has a change at a position in the predicted helix-turn-helix DNA binding structure which lies in a conserved region of the protein (region 4). This mutant specifically suppresses promoter down mutations in the -35 region of the promoter. The other mutant has a change at a residue that lies in a predicted α-helix of conserved region 2. This mutant specifically suppresses promoter mutations in the -10 region of the promoter. These data support the idea that regions 2 and 4 of sigma interact with the -10 and -35 regions of the promoter, respectively.

In this dissertation I describe the isolation and characterization of genomic clones for the human synapsin I gene, the establishment of a linkage map for the human synapsin I gene locus, and studies of the possible involvement of this gene in neurological disease. Synapsin I is a neuron-specific phosphoprotein which is concentrated at the presynaptic terminal. Evidence suggests that it plays a fundamental role in the regulation of neurotransmitter release. Altogether 27,500 bp of the human synapsin I gene have been isolated, and the gene structure has been partially determined. DNA sequence comparisons between human and rat genes show a high degree of conservation. Sequenced exons display an 87% identity to each other. The synapsin I genomic clones were employed in the search for a polymorphic marker. A compound (AC)n repeat located 1000 base pairs downstream from the human synapsin I gene and within the last intron of the A-raf-1 gene has been identified. DNA database comparisons of the sequences surrounding the repeat indicate that the synapsin I gene and the A-raf-1 gene lie immediately adjacent to each other, in opposite orientation. Polymerase chain reaction amplification of this synapsin I / A-raf-1 associated repeat using total genomic DNA from members of the 40 reference pedigree families of the Centre d'Etude du Polymorphisme Humaine showed it to be highly polymorphic, with a polymorphic information content value of 0.84 and a minimum of eight alleles. Because the synapsin I gene had been mapped previously to the short arm of the human X chromosome at Xp11.2, linkage analysis was performed with markers on the proximal short arm of the X chromosome. The most likely gene order is: DXS7 - SYN/ARAF1 - TIMP - DXS255 - DXS146 with a relative probability of 5 x 108 compared with the next most likely order. The SynI/Araf marker was next utilized in a linkage study aimed at establishing a more accurate placement of the genetic locus responsible for the ocular disorder Congenital stationary night blindness, which had been mapped previously close to DXS7. Our results confirm this prior localization and also exclude any placement proximal to the SYN/ARAF1 locus. Finally, the inheritance of the different alleles of the SynI/Araf marker in three families with Rett syndrome, a severe neurodegenerative disorder, which has been assigned to the X chromosome, was studied. In at least one of the families in which two half sisters with the same mother suffer from the disease, the inheritance of Rett syndrome was discordant with the inheritance of the same allele for the SynI/Araf marker. Thus, this highly informative repeat has proven already effective in the study of X-linked diseases and should serve as a valuable marker for disease loci mapped to the Xp11 region.

Proteins, which are T cell-dependent antigens, preferentially induce antibodies of the IgG1 class in mouse, whereas LPS, which is a T-independent antigen, preferentially induces IgG3 and IgG2b. Interaction between CD40 on B cells and CD40 ligand (CD40L) on T cells has been shown to mediate T cell contact help for B cell proliferation, differentiation and immunoglobulin isotype switching. In addition, it has been shown that membranes from activated T cells induce germline γ1 transcripts, and that CD40 signaling induces germline γ1 transcripts. These results indicate that T cell contact help mediated by CD40 ligand (CD40L)-CD40 interaction may contribute to this preferential IgG1 isotype selection in response to T-dependent antigens by inducing transcription of germline Ig γ1 transcripts. Here we show that signaling via CD40 increases expression of a transiently transfected luciferase reporter plasmid driven by the germline γ1 promoter in M12.4.1 B lymphoma cells. By linker scanning mutation analysis of the promoter, we have identified a CD40 responsive region (CD40RR) which is able to confer inducibility by CD40L to a minimal c-fos promoter. The CD40RR contains three NF-кB-binding sites, each of which is required for maximal induction of the γ1 promoter activity by CD40L. Binding of the NF-кB/Re1 proteins p50, Re1A, c-Re1 and Re1B to the CD40RR can be induced by CD40 signaling in M12.4.1 cells or in splenic B cells. Co-transfection of expression plasmids for p50 together with Re1A or Re1B, but not p50 alone or p50 and c-Re1, transactivates the CD40RR in transient transfection assays in M12.4.1 cells. These data demonstrate NFкB/Re1 proteins activated by CD40 engagement play an important role in regulation of the germline γ1 promoter. Further support for this conclusion is provided by the finding that treatment of splenic B cells with NF-кB inhibitors prevents induction of germline γ1 transcripts by CD40L. Although LPS also induces NF-кB activation, it poorly induces germline γ1 promoter activity in M12.4.1 cells and it also poorly induces germline γ1 transcripts in splenic B cells and in the mouse B cell line, 1B4.B6. Western blot analyses show that LPS predominantly activates p50 and c-Re1, whereas CD40L induces all NF-кB/Re1 proteins (Re1A, Re1B, c-Re1 and p50). Likewise, in nuclear extracts from LPS-treated cells, p50/cRe1 and p50/p50 dimers are the major NF-кB/Re1 proteins which bind to the promoter for germline γ1 transcripts in electrophoretic mobility shift assays, whereas in nuclear extracts from CD40L-treated cells, p50/Re1A and p50/Re1B dimers are the major complexes. Reporter gene assay by over expressing NF-кB/Re1 fusion proteins indicates that p50/Re1A and p50/Re1B dimers, but not p50/c-Re1 or p50/p50 dimer, can transactivate the germline γ1 promoter. Despite their inability to activate the promoter, p50/c-Re1 and p50/p50 can bind to the promoter and suppress the transactivation activity of p50/Re1A and p50/Re1B. Therefore, the effect of NF-кB activation on the germline γ1 promoter depends on the Relative amounts of transactivating and non-trans activating NF-кB/Re1 dimers. The inability of LPS to induce germline γ1 transcripts can be explained by induction of non-transactivating NF-кB/Re1 dimers and the ability of CD40L to activate the promoter by a greater induction of Re1A and Re1B Re1ative to c-Re1.