MHC class II regulatory factor RFX has a novel DNA-binding domain and a functionally independent dimerization domain.

The regulation of MHC class II gene expression controls T-cell activation and, hence, the immune response. Among the nuclear factors observed to bind to conserved DNA sequences in human leukocyte antigen (HLA) class II gene promoters, RFX is of special interest: Its binding is defective in congenital HLA class II deficiency, a disease of class II gene regulation. The cloning of an RFX cDNA has allowed us to show by transfection of a plasmid directing the synthesis of antisense RFX RNA that RFX is a class II gene regulatory factor. RFX is a novel 979-amino-acid DNA-binding protein that contains three structurally and functionally separate domains. The 91-amino-acid DNA-binding domain is distinct from other known DNA-binding motifs but may be distantly related to the helix-loop-helix motif. The most striking property of RFX is that it can bind stably to the class II X box as either a monomer or a homodimer and that the domain responsible for dimerization is distant from and functionally independent of the DNA-binding domain. This distinguishes RFX from other known dimeric DNA-binding proteins. It also implies that an RFX homodimer has two potential DNA-binding sites. We therefore speculate that RFX could form a DNA loop by cross-linking the two X-box sequences found far apart upstream of MHC class II genes.

Direct evidence for a functional role of HLA-DRB1 and -DRB3 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T lymphocytes.

The contribution of the HLA-DRB1, -B3, and -B5 gene products in the recognition of Dermatophagoides spp. (house dust mite) by helper T cells isolated from an atopic individual (HLA-DRw12, DR7; DRw52b) with perennial rhinitis was investigated. Using a panel of histocompatible and histoincompatible accessory cells, the restriction specificity obtained for a long term T cell suggested that a component of the dust mite reactive repertoire recognized antigen in association with DRB3 gene products. Oligonucleotide DNA typing of the presenting cell panel demonstrated a correlation between the DRw52b allele and T cell responsiveness. Murine fibroblasts expressing DRw52b, but not DRw52a or -c molecules, presented antigen to both the T cell line and cloned T cells (DE26) derived from the line, indicating that the supertypic specificity DRw52b was able to restrict recognition of dust mite antigens. Additional T cell clones (DE9 and DE41) also isolated from the line were restricted by the products of the B1 gene locus (DRw12B1) as determined by murine fibroblasts transfected with the appropriate HLA-DR genes. Clone DE9 was degenerate in its restriction specificity, also recognizing dust mite presented by accessory cells expressing the DR2 subtypes. Presentation by fibroblasts transfected with DRw12B1, DR2Dw2B5 genes and EBV-transformed B cell lines expressing DR2DW21B1 and -B5 indicated that the functional site restricting recognition may be associated with residues 70 and 71 of the DR beta chain helical wall of the antigen combining site. Furthermore, we have recently demonstrated that both T cell clones DE9 and DE26 induce allergen dependent IgE synthesis in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of HLA class II genes by IFN-gamma is transcriptional and requires a trans-acting protein.

HLA class II Ag are encoded by a family of related genes clustered in the HLA-D region of the MHC. The expression of this multi-gene family is highly regulated and this regulation is essential for the control of the immune response. Class II gene expression is constitutive in a limited number of cell types and can be induced by IFN-gamma in a number of class II negative cells. In this study, we have clarified two essential aspects of the regulation of HLA class II genes by IFN-gamma. 1) The induction mechanism operates at the level of transcription and there is a long lag phase in the signal transduction process. 2) The induction of class II genes requires the de novo synthesis of a new protein(s). On this basis, we propose that IFN-gamma regulates the transcription of HLA class II genes via the de novo synthesis of a trans-acting activator protein.

Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion.

HLA class II molecules were isolated from mouse L cells transfected with a DR alpha gene and an allele, 52a, of locus DR beta III from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described specificity TR22 encoded by another allele, 52b, of the DR beta III locus. The TR81 specificity was also present on the DR beta I gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DR beta genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DR beta III locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DR beta I gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DR beta chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DR beta I chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.

Alternative splicing and alternative initiation of translation explain the four forms of the Ia antigen-associated invariant chain.

The Ia antigen-associated invariant chain (In) exists in humans as four related polypeptides, p33, p35, p41 and p43, all associated with HLA-class II antigens. As described previously, two of these forms of In chain, p33 and p35, result from the use of two in-phase initiation AUG codons on the unique In p33 mRNA. In addition to cDNA clones derived from In p33 mRNA, we have isolated a new cDNA clone, called p41-1, which differs from p33-1 by an additional segment in the coding region. The DNA sequence encoding the segment unique to p41-1 was identified in the genomic sequence in the intron between exon 6 and 7, and we refer to it as exon 6b. Cells transfected with a full length p41 cDNA clone in an expression vector synthesize the two larger forms of the In chain, p41 and p43. We propose that the larger mRNA, encoding p41, results from alternative splicing of exon 6b, and that p41 and p43 result from the use of the two functional initiation AUG codons identified in p33 mRNA. Alternative splicing, together with alternative initiation of translation, allows therefore the synthesis of four related In chain polypeptides from a single gene.

Catalase anabolism in yeast: loss of regulation by oxygen of catalase apoprotein synthesis after mutation.

A mutant of Saccharomyces cerevisiae which displays catalase activity when grown under strictly anaerobic conditions has been selected on solid media. Although some preformed holoenzyme has accumulated in anaerobic cells, a sharp increase of activity is still measured during adaptation to oxygen in glucose-buffer; however, a striking difference with the wild-type strain is that in the mutant, catalase formation is observed in the presence of cycloheximide that totally inhibits cytoplasmic translation. It is concluded that kat 80 mutant has lost the regulatory control by oxygen of apocatalase synthesis; the later precursor, characterized as apocatalase synthesis; the latter precursor, characterized as apocatalase T, is thought to be activated in vivo, under aerobic conditions, by inclusion of prosthetic group. Regulation of enzyme synthesis by catabolite repression (glucose erfect) persists, unmodified by reference to the wild-type parental strain. Mutation kat 80 specifically hits catalase anabolism, as no significant variations were observed for the edification of the respiratory system and (apo)cytochrome c peroxidase production. Genetic analysis shows that kat 80 phenotype, recessive in heterozygotes, results from a single nuclear mutation.