Deletional analysis of the murine IL-12 p35 promoter comparing IFN-gamma and lipopolysaccharide stimulation.

IL-12, pivotal to the development of Th1 cells and formed by association of p35 and p40 subunits, is made by macrophages and the macrophage cell line RAW264.7. In this study, the promoter for p35 was cloned and analyzed. The murine IL-12 p35 gene has promoters upstream from each of the first two exons. The exon 1 and exon 2 promoters, cloned into a reporter vector, were responsive to LPS or IFN-gamma/CD40 ligation in transfected RAW264.7 cells. The exon 2 promoter containing bp -809 to +1 has significant homology to the human p35 promoter. Thus, deletion analysis was performed to determine the regions required for responsiveness to LPS, CD40, and/or IFN-gamma. Base pairs -809 to -740 influenced responsiveness to LPS. In contrast, bp -740to -444 and bp -122 to -100 were required for responses to IFN-gamma, IFN-gamma/LPS, or IFN-gamma/CD40 ligation. Removal of bp -444 to -392 increased the response of the exon 2 promoter to each stimulant. IFN regulatory factor (IRF)-1 is involved in the activity of this promoter at bp -108 to -103 because levels of nuclear IRF-1 correlated with exon 2 promoter activity in response to IFN-gamma and IRF-1 overexpression stimulated and enhanced exon 2 promoter activity. Also, site or deletion mutation of the IRF-1 element at bp -108 to -103 reduced the responsiveness of the promoter and IRF-1 bound to an oligonucleotide containing bp -108 to -103. The data suggest that the response of the p35 promoter to IFN-gamma requires a distinct IRF-1 positive regulatory element at bp -108 to -103.

Phanerochaete chrysosporium glyoxal oxidase is encoded by two allelic variants: structure, genomic organization, and heterologous expression of glx1 and glx2.

A cDNA clone (glx-2c) encoding glyoxal oxidase (GLOX) was isolated from a Phanerochaete chrysosporium lambda gt11 library, and its nucleotide sequence was shown to be distinct from that of the previously described clone glx-1c (P. J. Kersten and D. Cullen, Proc. Natl. Acad. Sci. USA 90:7411-7413, 1993). Genomic clones corresponding to both cDNAs were also isolated and sequenced. overall nucleotide sequence identity was 98%, and the predicted proteins differed by a single residue: Lys-308<==>Thr-308. Analyses of parental dikaryotic strain BKM-F-1767 and homokaryotic progeny firmly established allelism for these structural variants. Southern blots of pulsed-field gels localized the GLOX gene (glx) to a dimorphic chromosome separate from the peroxidase and cellobiohydrolase genes of P. chrysosporium. Controlled expression of active GLOX was obtained from Aspergillus nidulans transformants when glx-1c was fused to the promoter and secretion signal of the A. niger glucoamylase gene. The GLOX isozyme corresponding to glx-2c was also efficiently secreted by A. nidulans following site-specific mutagenesis of the expression vector at codon 308 of glx-1c.

Clonal expansion and persistence of human T cells specific for an immunodominant myelin basic protein peptide.

TCR rearrangements were used to probe the clonal origin of myelin basic protein (MBP)-reactive T cells from patients with multiple sclerosis (n = 7) and normal subjects (n = 3). The majority of MBP-specific T cell lines were specific for the immunodominant MBP(84-102) and MBP(143-168) peptides and were restricted by HLA-DR molecules. In two patients with the DR2 haplotype, the T cell response to MBP was focused on the MBP(84-102) peptide. In both patients, in vivo expanded population(s) (three expanded populations in the first patient, one expanded population in the second patient) dominated the response to the MBP(84-102) peptide. Two MBP(84-102)-specific T cell clones from a normal subject with the DR2 haplotype were also found to have identical TCR sequences. Clonality was proven by demonstrating that independent clones had identical TCR alpha- and TCR beta-chain sequences as well as identical sequences of a TCR gamma-chain or of a second TCR alpha-chain rearrangement. Repeated analysis of one patient after 13 mo demonstrated that the three expanded clones had persisted in vivo. A representative of one of the expanded clones was again obtained after 31 mo by IL-2 stimulation suggesting that this clone was activated in vivo. These data suggest that the response to human MBP is dominated in at least some subjects by expanded clones that may persist in vivo for relatively long periods of time.