Immunization with the Sm nuclear antigen induces anti-Sm antibodies in normal and MRL mice.

The spontaneous occurrence of antibodies against the Sm nuclear antigen is a highly specific marker for the diagnosis of SLE. We have previously shown that anti-Sm can be elicited by immunization of SLE-prone mice with purified Sm antigen. In the present study, this autoantibody was induced in normal mice by a similar immunization protocol. Anti-Sm produced by normal strains was predominantly IgG1, which is similar to the isotype distribution in Sm-immunized MRL mice, but unlike the IgG2a-dominated response seen for spontaneous anti-Sm. Anti-Sm raised by immunization in most strains recognized epitopes not seen by spontaneous human and murine SLE anti-Sm; of the eleven normal strains tested, only C3H and AKR, strains from which MRL was partially derived, responded to these determinants. Further, immunoblot analysis of anti-Sm generated by immunization of MRL and normal mice revealed that the same proteins recognized by spontaneous human and murine anti-Sm were also seen by these sera. This study shows that an autoantibody highly characteristic of SLE can be produced in normal and MRL mice after appropriate immunization, and that the fine specificity of such experimentally induced antibody can be similar to that of spontaneous anti-Sm autoantibodies. The results imply a role for autoimmunization with Sm in the production of anti-Sm.     

Citrus exocortis viroid RNA is associated with the largest subunit of RNA polymerase II in tomato in vivo

Summary.  An active replication complex of citrus exocortis viroid (CEV) was isolated as a chromatin-enriched fraction of infected tomato leaf with CEV RNA synthesis activity. This activity was solubilised from the chromatin with ammonium sulphate, but not with sarkosyl. Nucleoprotein complexes in the soluble fraction which bound to a monoclonal antibody to the carboxy terminal domain of the largest subunit of RNA polymerase II (8WG16) were affinity purified and contained plus- and minus-sense CEV RNA. The results support a role for RNA polymerase II in viroid replication and provide the first direct evidence of an association in vivo between host RNA polymerase II and CEV. Received March 28, 1999/Accepted July 21, 1999     

Chromosomal assignment of gene encoding the largest subunit of RNA polymerase II in the mouse

The gene encoding the largest subunit of RNA polymerase II was mapped to mouse chromosome 11 by Southern blotting analysis of mouse-Chinese hamster somatic cell hybrids and by in situ hybridization. This assignment extends the previously defined homology between mouse chromosome 11 and human chromosome 17.     

Characterization of the gene encoding the largest subunit of Plasmodium falciparum RNA polymerase III.

We report here the isolation, sequence analysis, structure, and expression of the gene encoding the largest subunit of RNA polymerase III (RPIII) from Plasmodium falciparum. The P. falciparum RPIII gene consists of 5 exons and 4 introns, is expressed in all of the asexual erythrocytic stages of the parasite as a 8.5-kb mRNA, and is present in a single copy on chromosome 13. The predicted 2339 amino acid residue RPIII subunit contained 5 regions that were conserved between different eukaryotic RPIII subunits, and 4 variable regions that separated the conserved regions. Three of the variable regions were greatly enlarged in comparison to the corresponding variable regions in other RPIII subunits. Variable region C' represented nearly one-third of the P. falciparum RPIII subunit (750 amino acid residues), included a unique repeated decapeptide sequence, and had some homology with yeast DNA topoisomerase II. Noteworthy amino acid sequences and structures were identified in both the conserved regions and in the enlarged variable regions, and their possible role(s) as domains that regulate RPIII enzyme activity is discussed.     

Expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) in the mouse can elicit the production of anti-dsDNA and anti-Sm antibodies

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by the production of anti-nuclear antibodies. The etiology of SLE is unknown, although several viruses including the Epstein-Barr virus (EBV) have been implicated. An increase in the frequency of EBV infection has been observed in SLE patients relative to normal individuals. Some patients with SLE develop antibodies that recognize a proline rich epitope in the ribonucleoprotein Sm B/B that is similar to an epitope in EBNA-1, a major nuclear antigen of EBV. In the present study we have cloned the EBNA-1 gene under the control of the CMV promoter in the vector pcDNA3. We now report for the first time that expression of the entire EBNA-1 protein in the mouse can elicit the production of IgG antibodies to Sm and to double-stranded DNA (dsDNA). Our data suggest that the anti-Sm response arises as a consequence of antigenic cross-reactivity by anti-EBNA-1 antibodies. These results support a possible association between EBV infection and SLE.     

Purification of an eight subunit RNA polymerase I complex in Trypanosoma brucei

Trypanosoma brucei harbors a unique multifunctional RNA polymerase (pol) I which transcribes, in addition to ribosomal RNA genes, the gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. In consequence, this RNA pol I is recruited to three structurally different types of promoters and sequestered to two distinct nuclear locations, namely the nucleolus and the expression site body. This versatility may require parasite-specific protein-protein interactions, subunits or subunit domains. Thus far, data mining of trypanosomatid genomes have revealed 13 potential RNA pol I subunits which include two paralogous sets of RPB5, RPB6, and RPB10. Here, we analyzed a cDNA library prepared from procyclic insect form T. brucei and found that all 13 candidate subunits are co-expressed. Moreover, we PTP-tagged the largest subunit TbRPA1, tandem affinity-purified the enzyme complex to homogeneity, and determined its subunit composition. In addition to the already known subunits RPA1, RPA2, RPC40, 1RPB5, and RPA12, the complex contained RPC19, RPB8, and 1RPB10. Finally, to evaluate the absence of RPB6 in our purifications, we used a combination of epitope-tagging and reciprocal coimmunoprecipitation to demonstrate that 1RPB6 but not 2RPB6 binds to RNA pol I albeit in an unstable manner. Collectively, our data strongly suggest that T. brucei RNA pol I binds a distinct set of the RPB5, RPB6, and RPB10 paralogs.     

Effect of the xid gene on graft-versus-host-induced autoantibody production in nonautoimmune mice

Graft-versus-host disease (GVH) was used to induce an autoimmune state in F1 recipients using donor spleen cells, splenic T cells, or Lyt 1+2- splenic T cells from either normal DBA/2 mice or from DBA/2 mice carrying the X-linked immunodeficiency (xid) gene. Recipients were either nondefective (DBA/2 X CBA/N)F1 males or reciprocal cross (CBA/N X DBA/2)F1 male mice carrying the xid gene. GVH induced hypergammaglobulinemia and anti-ssDNA autoantibodies in F1 recipients. Immunodeficient (CBA/N X DBA/2)F1 recipients had less hypergammaglobulinemia and IgG anti-ssDNA than did normal (DBA/2 X CBA/N)F1 recipients. Spleen cells, splenic T cells, and Lyt 1+2- splenic T cells from immunodeficient DBA/2.xid donors were less able to induce GVH and autoimmunity than normal DBA/2 donors. These studies suggest that the xid gene may reduce the autoimmune hyperractive state, but may do so by acting on more than one cell population, including T cells.     

Fine structure mapping and phenotypic analysis of five temperature-sensitive mutations in the second largest subunit of vaccinia virus DNA-dependent RNA polymerase

We have used plasmid clones spanning the region encoding the 132-kDa subunit of the cowpox virus RNA polymerase (CPV rpo 132) to marker rescue each of five vaccinia virus (VV) temperature sensitive (ts) mutants, ts 27, ts 29, ts 32, ts 47, and ts 62, which together constitute a single complementation group. The experiments fine-map the vaccinia mutations to a 1.3-kb region containing the 3' end of the CPV rpo 132 gene. Phenotypic characterization shows that all five mutants are affected to varying extents in their ability to synthesize late viral proteins at the nonpermissive temperature, similar to other ts mutants with lesions in the 22- and the 147-kDa subunits of the VV RNA polymerase. Two mutants, ts 27 and ts 32, exhibit a delay in the synthesis of late viral proteins at both the permissive and the nonpermissive temperatures. We conclude that the five VV mutants affect the 132-kDa subunit of the VV RNA polymerase. Additional genetic experiments demonstrate intragenic complementation between ts 62 and three other members of this complementation group, ts 27, ts 29, and ts 32.     

Ubiquitous expression and embryonic requirement for RNA polymerase II coactivator subunit Srb7 in mice

Mammalian RNA polymerase II complexes and coactivators containing homologs of yeast Srb/Med proteins have been isolated recently from tissue culture cells. The yeast Srb/Med complex is involved in global gene expression and is essential, but it is not yet known if its mammalian counterparts are broadly expressed in tissues or if they are essential. We have isolated the murine gene encoding Srb7, an Srb/Med complex protein whose sequence and function is highly conserved between yeast and humans. The mouse Srb7 gene is single copy, and Northern analysis showed that it is expressed in all tissues examined. Disruption of the gene in embryonic stem cells revealed that it is essential for cell viability and murine embryonic development. These results, together with evidence that murine Srb7 is associated exclusively with high molecular weight forms of RNA polymerase II in extracts, suggest that Srb7-containing polymerase complexes occur in most tissues and have essential roles in expression of protein coding genes.     

Immunization of BALB/c mice with pigeon IgY induces the production of anti-IgG autoantibodies

The breakdown of immunological tolerance due to the activation of autoreactive B and T cells triggers physiopathological processes. An example of such conditions is the production of IgG autoantibodies specific for the Fc portion of IgG (anti-Fcγ IgG). Previous reports have shown that patients with pigeon-related hypersensitivity pneumonitis exhibit an increase in the serum levels of anti-Fcγ IgG. There is no in vivo model for the study of this condition and the immunological mechanisms of tolerance breakdown associated with sensitization by pigeon antigens are still unknown. In this work, we show that the repeated immunization of BALB/c mice with pigeon IgY during 16-weeks induces the production of anti-Fcγ IgG and keeps their high levels for seven weeks. The late appearance of anti-Fcγ IgG autoantibodies in the plasma is similar to what has been reported in other experimental autoimmune models. With the occurrence of anti-Fcγ IgG, there is a reduction in the proportion of Foxp3?+?cells (regulatory T cells, Tregs) within the population of splenic CD4?+?CD25?+?T cells. Thus, our data showed that the immunization of BALB/c mice with IgY promotes the production of anti-Fcγ IgG along with a decrease in Tregs in the spleen. We propose that immunization of mice with pigeon antigens, like IgY can provide a model to study the immunological mechanisms involved in the development of pigeon-related hypersensitivity pneumonitis.     

Mutations in conserved domains IV and VI of the large (L) subunit of the sendai virus RNA polymerase give a spectrum of defective RNA synthesis phenotypes

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein. When aligned with amino acid sequences of L proteins from other negative-sense RNA viruses, the Sendai L protein contains six regions of good conservation, designated domains I-VI, which have been postulated to be important for the various enzymatic activities of the polymerase. To directly address the roles of domains IV and VI, 14 site-directed mutations were constructed either by changing clustered charged amino acids to ala or by substituting selected Sendai L amino acids with the corresponding sequence from measles virus L. Each mutant L protein was tested for its ability to transcribe and replicate the Sendai genome. The series of mutations created a spectrum of phenotypes, from those with significant, near wild-type, activity to those being completely defective for all RNA synthesis. The inactive L proteins, however, were still able to bind P protein and form a polymerase capable of binding the nucleocapsid template. The remainder of the mutations reduced, but did not abolish, enzymatic activity and included one mutant with a specific defect in the synthesis of the leader RNA compared with mRNA, and three mutants that replicated genome RNA much more efficiently in vivo than in vitro. Together, these data suggest that even within a domain, the function of the Sendai L protein is likely to be very complex. In addition, SS3 and SS10 L in domain IV and SS13 L in domain VI were shown to be temperature-sensitive. Both SS3 and SS10 gave significant, although not wild-type, activity at 32 degrees C; however, each was completely inactivated for all RNA synthesis at 37 and 39.6 degrees C. SS13 was completely inactive only when synthesized at the higher temperature. Each polymerase synthesized at 32 degrees C could only be partially heat inactivated in vitro at 39.6 degrees C, suggesting that inactivation involves both thermal lability of the protein and temperature sensitivity for its synthesis.     

Blockade of TLR9 signaling in B cells impaired anti-dsDNA antibody production in mice induced by activated syngenic lymphocyte-derived DNA immunization

We previously established a systemic lupus erythematosus (SLE) animal model in non-susceptible BALB/c mice by immunizing with activated syngeneic lymphocyte-derived DNA (ALD-DNA), manifested by high level of anti-double-stranded DNA (dsDNA) antibodies (Abs), proteinuria, glomerular deposition of immune complex and glomerulonephritis. The production of anti-dsDNA Abs is closely related with the renal inflammation and damage in this model. However, recognition of ALD-DNA and its signaling pathway within antigen-presenting cells (APC) remains not fully clarified. Herein, in this study, Toll-like receptor 9 (TLR9), a well-known pattern-recognition receptor for dsDNA with CpG motif, was found to be dynamically up-regulated in B cells during the process of the SLE disease. Knockdown of TLR9 by short interfering RNA (siRNA) in B cells in vitro and in vivo reduced the production of anti-dsDNA antibody and consequently ameliorated the SLE syndrome in mice while the affinity and isotype of the antibody remained the same. Our results implied that TLR9 signaling of B cells might play an important role in the production of anti-dsDNA Abs triggered by auto dsDNA, which would extend our understanding of TLR9 immune recognition in the pathogenesis of SLE disease.