Production of interleukin-12 as a self-processing 2A polypeptide.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of two disulfide-linked subunits (p40 and p35) encoded by separate genes. We used the apparent autocleavage property of a 2A peptide from the foot-and-mouth disease virus (FMDV) to express bovine (Bo) IL-12 as a self-processing polypeptide (p402Ap35). We demonstrate that 2A will mediate the cleavage of p402Ap35 into two separate subunits in a manner similar to that observed during the processing of the FMDV polypeptide. Furthermore, this 2A polypeptide encoded a functional heterodimer, which elicited activities associated with IL-12 in other species. We propose that this strategy of self-processing polypeptides may be used in many applications where the coordinated and stoichiometric expression of complex proteins is required.

Influence of IL-12 on interferon-gamma production by bovine leucocyte subsets in response to bovine respiratory syncytial virus.

The cytokine IL-12 is a key molecule in the regulation of CD4+ T cell development and specifically potentiates the development of T helper 1 responses in mouse and man. However the biological effects mediated by bovine IL-12 have not been defined in cattle. To produce the expression of the two mature proteins a polyprotein approach was used. This system is employed by positive strand viruses and encodes both products from a single open reading frame (ORF). The 2A region of foot-and-mouth disease virus (FMDV) encodes a site that appears to undergo auto-cleavage. Here the 2A was flanked by sequences encoding the p35 and p40 polypeptides of the heterodimeric cytokine to mediate their cleavage. Formation of the correct heterodimeric structure is an absolute requirement for IL-12 biologic activity. Using bovine respiratory syncytial virus (BRSV) and ovalbumin (OVA) we studied the effects of IL-12 on the responses of peripheral blood mononuclear cells (PBMC) to these antigens, in vitro. The presence of IL-12 markedly influenced the level of IFNg secreted by these cells, and although IL-12 induced IFNg production in the absence of antigenic stimulation, IFNg production was accelerated and augmented in response to IL-12 and antigen. Analysis of the T cell subsets by flow cytometry showed that CD4+ T cells comprised the largest contributors to IFNg production. The WC1 + gd T cells did not appear to contribute to the production of IFNg.

EMBL-Align: a new public nucleotide and amino acid multiple sequence alignment database.

UNLABELLED: The submission of multiple sequence alignment data to EMBL has grown 30-fold in the past 10 years, creating a problem of archiving them. The EBI has developed a new public database of multiple sequence alignments called EMBL-Align. It has a dedicated web-based submission tool, Webin-Align. Together they represent a comprehensive data management solution for alignment data. Webin-Align accepts all the common alignment formats and can display data in CLUSTALW format as well as a new standard EMBL-Align flat file format. The alignments are stored in the EMBL-Align database and can be queried from the EBI SRS (Sequence Retrieval System) server. AVAILABILITY: Webin-Align: http://www.ebi.ac.uk/embl/Submission/align_top.html, EMBL-Align: ftp://ftp.ebi.ac.uk/pub/databases/embl/align, http://srs.ebi.ac.uk/