Transcription, beta-like DNA polymerases and hypermutation.

This paper discusses two aspects of immunoglobulin (Ig) gene hypermutation. In the first approach, a transcription termination signal is introduced in an Ig light chain transgene acting as a mutation substrate, and transgenic lines are generated with control and mutant transgenes integrated in tandem. Analysis of transcription levels and mutation frequencies between mutant and control transgenes clearly dissociates transcription elongation and mutation, and therefore argues against models whereby specific pausing of the RNA polymerase during V gene transcription would trigger an error-prone repair process. The second part reports the identification of two novel beta-like DNA polymerases named Pol lambda and Pol mu, one of which (Pol mu) represents a good candidate for the Ig mutase due to its higher lymphoid expression and its similarity with the lymphoid enzyme terminal deoxynucleotidyl transferase. Peculiar features of the expression of this gene, including an unusual splicing variability and a splicing inhibition in response to DNA-damaging agents, are discussed.

Monitoring gene expression of TNFR family members by beta-cells during development of autoimmune diabetes.

Autoimmune diabetes results from destruction of pancreatic beta-cells by islet-infiltrating leukocytes. Different molecular mechanisms seem to be involved in this destruction but the results from many studies have not provided a clear picture so far. Therefore, we have developed a multiplex single-cell reverse transcription polymerase chain reaction to analyze the expression of genes of the tumor necrosis factor receptor (TNFR) family in pancreatic beta-cells during the development of autoimmune diabetes in a TCR-HA x INS-HA double transgenic as well as a non-obese diabetic (NOD) animal model. To this end we have followed the expression of cell surface receptors of the TNFR family in NOD mice as well as in double transgenic mice that express in their T cells class II MHC-restricted TCR specific for peptide 111 - 119 from influenza hemagglutinin (TCR-HA) as well as hemagglutinin under the control of the rat insulin promoter (INS-HA). Both types of mice develop insulitis and diabetes spontaneously. The data show a significant increase in the expression of Fas and TNFR2 (p75) during the development of insulitis, whereas TNFR1 (p55) is already expressed in beta-cells before the onset of insulitis. As ligands for these receptors are already expressed at high levels during the phase of insulitis, it is possible that beta-cell death is regulated by intracellular inhibitors of apoptosis pathways.

Crucial function of the pre-T-cell receptor (TCR) in TCR beta selection, TCR beta allelic exclusion and alpha beta versus gamma delta lineage commitment.

The analysis of T-cell receptor (TCR) beta selection, TCR beta allelic exclusion and TCR beta rearrangement in gamma delta T cells from normal and pre-TCR-deficient mice has shown that the pre-TCR has a crucial role in T-lymphocyte development: The pre-TCR is by far the most effective receptor that generates large numbers of CD4+8+ T cells with productive TCR beta rearrangements. In the absence of the pre-TCR, TCR beta rearrangement proceeds in developing cells irrespective of whether they already contain a productive TCR beta gene. The pre-TCR directs developing T cells to the alpha beta lineage because gamma delta T cells from pT alpha-/- mice proceed much further in TCR beta rearrangement than gamma delta T cells from wild-type mice. It is argued that the pre-TCR commits developing T cells to the alpha beta lineage by an instructive mechanism, which has largely replaced an evolutionarily more ancient mechanism that involves stochastic alpha beta lineage commitment.