No abstract

Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a "split and inverted" configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line ⑀ transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides.

A deletion event in the T cell receptor (TcR) delta locus has been characterized in a panel of mouse alpha/beta cytotoxic T lymphocyte (CTL) clones. Data presented here shows that J delta 1, J delta 2 and C delta are absent from functional CTL clones while a germ-line D delta 1 fragment is retained, thus suggesting a specific deletion of this region. We have investigated the possible significance of the J-C delta deletion by generating T cell lines from TcR alpha/beta transgenic mice. Unlike control T cell lines which included a T cell line derived from a beta transgenic mouse, the lines expressing the transgenic alpha/beta heterodimer have not deleted the C delta region. This strongly suggests that the J-C delta deletion event is not responsible for directing T cells to the alpha/beta lineage, but rather is involved in the rearrangement or transcriptional activity of the alpha locus. In addition, to ensure that the alpha/beta transgene does not have any inhibitory affects on the rearrangement of the delta loci in general, the gamma/delta expressing dendritic epithelial T cell (DETC) population was examined in TcR alpha/beta transgenic mice and alterations in this T cell subset were not found. This finding that normal gamma/delta DETC cells are present in alpha/beta transgenic mice, together with the data showing that the D delta 1 region remains in an unrearranged germ-line configuration in functional alpha/beta CTL, suggests that commitment to the alpha/beta or gamma/delta lineage is predetermined at a particular stage in early T cell ontogeny.