We have produced transgenic mice expressing hen egg-white lysozyme (HEL) under the control of a ubiquitous promoter, so that in transgenic animals, HEL is presumably present in the serum and thymus throughout the period of establishment of the T-cell repertoire. We show that HEL transgenic H-2d mice with HEL blood levels >10 ng/ml are tolerant to HEL as well as to the immunodominant peptide 108-116. Thus, their T lymphocytes do not proliferate in response to the immunodominant peptide 108-116 after in vivo immunization with HEL or peptide 108-116. In contrast, in transgenic mice tolerant to HEL, the state of tolerance to subdominant peptides 1-18 and 74-96 appears variable and highly dependent on HEL blood levels. Complete unresponsiveness is seen when HEL serum levels are high, and this unresponsiveness is reached at a lower HEL concentration for peptide 1-18 than for peptide 74-96. Thus, a hierarchy exists among the three peptides (108-116 >> 1-18 > 74-96) for induction of a response to HEL and for HEL tolerance induction in T cells specific for these peptides. Persistence in the periphery of autoreactive T cells recogiing subdominant peptides of self-proteins, as shown in this transgenic model, indicates that self-tolerance is limited to a subset of dominant self-peptides and suggests a role for T lymphocytes specific for subdominant determinants in autoimmunity.

SummaryWe have previously produced a transgenic mouse line for hen egg lysozyme (HEL), an experimental model for analyzing tolerance to self-antigens at the peptide level. We have now characterized transgenic mice with HEL blood levels below 2 ng/ml, where significant T cell proliferative responses to HEL and its immunodominant peptide were observed. This HEL-low transgenic model was chosen because it mimics physiological conditions in which autoreactive T lymphocytes, recognizing self-components expressed at very low levels, persist without inducing a break in tolerance. Furthermore, in H-2 d mice, HEL-specific T lymphocytes are triggered by a single immunodominant region, allowing us to compare the HEL-specific T cell VB repertoires of transgenic and nontransgenic animals against a single peptide presented as self or foreign, respectively. We found that a V/38.2-D/31-J/31.5 rearrangement is found in response to HEL in all nontransgenic mice, whereas this V/3-restricted response is absent in HEL-low transgenic animals. At the nucleotide level, this rearrangement results from the trimming of the genomic segments during VDJ or DJjoining, without N additions, suggesting that the dominant rearrangement is selected early during fetal or neonatal life, before the expression of terminal deoxynucleotidyl transferase. In HEL-low transgenic mice, no dominant rearrangements are found as alternatives to the one observed in normal mice. Instead, each transgenic animal uses a different set of V~-J/3 combinations in its response to the immunodominant HEL peptide. In nontransgenic mice, besides the dominant VB8.2-D/31-J~l.5 combination, minor VB repertoires were found which differed in each animal and were distinct from the rearrangements used by individual transgenic mice. These findings suggest that the T cell response to an immunodominant peptide involves a "public" V/5 repertoire found in all animals and a "private" one which is specific to each individual.

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.

The contribution of template-independent nucleotide addition to antigen receptor diversity is unknown. We therefore determined the size of the T cell receptor (TCR)α/β repertoire in mice bearing a null mutation on both alleles of the terminal deoxynucleotidyl transferase (Tdt) gene. We used a method based upon polymerase chain reaction amplification and exhaustive sequencing of various AV-AJ and BV-BJ combinations. In both wild-type and Tdt°/° mice, TCRAV diversity is one order of magnitude lower than the TCRBV diversity. In Tdt°/° animals, TCRBV chain diversity is reduced 10-fold compared with wild-type mice. In addition, in Tdt°/° mice, one BV chain can associate with three to four AV chains as in wild-type mice. The α/β repertoire size in Tdt°/° mice is estimated to be 105 distinct receptors, ∼5–10% of that calculated for wild-type mice. Thus, while Tdt activity is not involved in the combinatorial diversity resulting from α/β pairing, it contributes to at least 90% of TCRα/β diversity.

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The docking and fusion of synaptic vesicles with the presynaptic plasma membrane require the interaction of the vesicle-associated membrane protein VAMP with the plasma membrane proteins syntaxin and SNAP-25. Both of these proteins behave as integral membrane proteins, although they are unusual in that they insert into membranes post-translationally. Whereas VAMP and syntaxin possess hydrophobic transmembrane domains, SNAP-25 does not, and it is widely believed that SNAP-25 traffics to and inserts into membranes by posttranslational palmitoylation. In pulse-chase biosynthesis studies, we now show that SNAP-25 and syntaxin rapidly bind to each other while still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractionation studies revealed that cytosolic SNAP25⅐syntaxin complexes then traffic to and insert into membranes. Furthermore, the association of SNAP-25 with membranes is dramatically enhanced by syntaxin, and the transmembrane domain of syntaxin is essential for this effect. Surprisingly, despite the importance of the SNAP-25 palmitoylation domain for membrane anchoring at steady state, removal of this domain did not inhibit the initial association of newly synthesized SNAP-25 with membranes in the presence of syntaxin. These data demonstrate that the initial attachment of newly synthesized SNAP-25 to membranes is a consequence of its association with syntaxin and that it is only after syntaxin-mediated membrane tethering that SNAP-25 is palmitoylated.Calcium-stimulated exocytosis of synaptic vesicles is essential for the development of the central nervous system as well as the maintenance of proper neural signaling. For this reason, investigation of the proteins regulating synaptic vesicle docking and fusion with presynaptic plasma membranes is essential for a complete understanding of the mechanisms regulating this complex process. Through a combination of genetic and biochemical approaches, members of the SNARE family of proteins have been strongly implicated in the processes of vesicle docking and fusion (reviewed in Refs. 1-3). On the synaptic vesicle itself is a member of the vesicle SNARE family of VAMP-like proteins, whereas on the presynaptic plasma membrane, there are the target SNARE (t-SNARE) 1 proteins syntaxin and SNAP-25. To form a functional t-SNARE complex, syntaxin must first associate with SNAP-25 (4). The binding of syntaxin to SNAP-25 induces a conformational change in SNAP-25 (5), and it has been demonstrated that a preformed t-SNARE complex is necessary for the subsequent formation of the complete vesicle SNARE⅐t-SNARE complex (6). Following SNARE complex assembly, a poorly understood series of protein-protein, protein-lipid, and lipid-lipid interactions occur, with the net result of all of these interactions being the fusion of the opposing membranes and the release of neurotransmitter into the synaptic cleft. The t-SNARE syntaxin and the vesicle SNARE VAMP are carboxyl terminus-anchored transmembrane proteins with typical hydrophobic transmembrane domains....

T cell repertoires observed in response to immunodominant and subdominant peptides include private, i.e., specific for each individual, as well as public, i.e., common to all mice or humans of the same MHC haplotype, Vα-Jα and Vβ-Dβ-Jβ rearrangements. To measure the impact of N-region diversity on public repertoires, we have characterized the αβ TCRs specific for several CD4 or CD8 epitopes of wild-type mice and of mice deficient in the enzyme TdT. We find that V, (D), J usage identified in public repertoires is strikingly conserved in TdT°/° mice, even for the CDR3 loops which are shorter than those found in TdT+/+ animals. Moreover, the 10- to 20-fold decrease in αβ T cell diversity in TdT°/° mice did not prevent T cells from undergoing affinity maturation during secondary responses. A comparison of the CDR3β in published public and private repertoires indicates significantly reduced N-region diversity in public CDR3β. We interpret our findings as suggesting that public repertoires are produced more efficiently than private ones by the recombination machinery. Alternatively, selection may be biased in favor of public repertoires in the context of the interactions between TCR and MHC peptide complexes and we hypothesize that MHCα helices are involved in the selection of public repertoires.

SNAP-25 is a key protein required for the fusion of synaptic vesicles with the plasma membrane during exocytosis. This study establishes that SNAP-25 is di¡erentially phosphorylated by protein kinase C and protein kinase A in neuroendocrine PC12 cells. Using phosphopeptide mapping and site-directed mutagenesis we identi¢ed both Thr138 and Ser187 as the targets of SNAP-25 phosphorylation by protein kinase C and Thr138 as the exclusive site of SNAP-25 phosphorylation by protein kinase A in vivo. Finally, despite published data to the contrary, we demonstrate that stimulation of regulated exocytosis under physiological conditions is independent of a measurable increase in SNAP-25 phosphorylation in PC12 cells.