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W Reith

Publications and source records attributed to W Reith.

At least 91 records · Page 5Linked to original sources
PubMed1992

The genes for MHC class II regulatory factors RFX1 and RFX2 are located on the short arm of chromosome 19.

RFX1 is a transacting DNA-binding regulatory factor involved in the control of MHC class II gene expression. RFX2 is a structurally very similar protein with identical DNA binding features. A member of the family of RFX factors is affected in an autosomal recessive disease, MHC class II deficient combined immunodeficiency (CID), caused by a defect in a trans-acting regulatory factor controlling MHC class II gene expression. In situ hybridization with 3H-labeled RFX1 cDNA has allowed us to identify two distinct targets on the short arm of chromosome 19 (19p13.1 and 19p13.2-p13.3). With the use of biotinylated genomic cosmid clones specific for RFX1 and RFX2, respectively, it was then possible to localize RFX1 at 19p13.1 and RFX2 at 19p13.2-p13.3. These two regulatory genes are thus assigned to a region of high gene density and RFX1 is close to another DNA-binding factor, LYL1.

Chromosome Mapping↗
PubMed1992

The DNA-binding defect observed in major histocompatibility complex class II regulatory mutants concerns only one member of a family of complexes binding to the X boxes of class II promoters.

The X box of major histocompatibility complex class II promoters is essential for proper expression of class II genes. Here we show that two distinct protein-DNA complexes (A and B), which exhibit similar binding characteristics and identical contact points on the X box, can be formed. This suggests the existence of a family of related X box-binding factors. Complex B (and not complex A) is specifically affected in primary combined immunodeficiency, a congenital defect in class II gene regulation. RFX1, the first X box-binding protein cloned, encodes a functionally relevant factor present in complex A and not in complex B as originally suspected. This report also illustrates the need for caution in correlating specific cloned proteins with nuclear factors identified by DNA-binding assays, particularly when dealing with families of related proteins.

Base Sequence↗
PubMed1991

Regulatory factor-X binding to mutant HLA-DRA promoter sequences.

The class II genes of the major histocompatibility complex (MHC) encode a family of cell surface glycoproteins that present processed antigen to the T cell receptor. Class II genes are regulated coordinately, responding to both immunologic and developmental signals. Conserved sequence elements 5' to class II genes have been shown to be important in transcriptional control. One of these sequences, the X box, is a specific target for the binding of the factor RF-X. In the hereditary HLA class II deficiency, a form of primary immunodeficiency, a regulatory defect in expression of class II genes is associated with a defect in the binding of RF-X. To determine the basepairs that are important for this binding interaction, a series of single basepair substitutions spanning the X box motif of the DRA gene was constructed and tested for binding of RF-X by gel electrophoresis mobility shift assays (EMSAs). Several, but not all, of the mutants severely affected binding of RF-X. In addition, the binding of both the natural and the recombinant form of RF-X was affected with the same specificity. A comparison of X box basepair positions important for RF-X binding to DRA with sequences conserved between X boxes of other class II alpha chain genes suggests that high affinity RF-X binding is important for a high level of expression and may explain differences in the levels of class II expression of different class II alpha chains.

Animals↗
PubMed1991

Hyperexpression of interferon-gamma-induced MHC class II genes associated with reorganization of the cytoskeleton.

Class I and class II major histocompatibility complex (MHC) gene products are key recognition units in the induction and regulation of the immune response. Expression of class I and class II may be constitutive or inducible by cytokines such as interferon-gamma (IFN-gamma). A key step in the induction of MHC genes is recognition of IFN-gamma by its membrane receptor. The work described here examines the regulation of the occupied IFN-gamma receptor by the cytoskeleton. To do this the authors have used the fungal metabolites dihydrocytochalasin B (DHCB) and cytochalasin D (CD), substances that bind to actin filaments and thereby disrupt the cytoskeleton. The authors have studied the effect of DHCB and CD on IFN-gamma-induced MHC gene expression in 143 B cells, a human osteosarcoma-derived cell line. Herein the authors demonstrate that alterations in the cytoskeleton induced by DHCB and CD can lead to increases in IFN-gamma-induced MHC gene expression. Dihydrocytochalasin B added up to 3 hours after IFN-gamma results in a threefold to sixfold increase in levels of class II mRNA while producing minimal enhancement of class I gene expression. In contrast, glyceraldehyde-3-phosphate dehydrogenase mRNA expression was unaltered by IFN-gamma or by the cytochalasins. The increased amount of class II mRNA can be accounted for by a concomitant increase in transcription rate of this gene. Studies using 125I-IFN-gamma demonstrate that the occupied IFN-gamma receptor associates with a Triton X-100 insoluble fraction of 143 B cells and that DHCB and CD markedly inhibit this association. The results described here provide evidence that is consistent with the hypothesis that the activity of the occupied IFN-gamma receptor may be modulated by interactions with the cytoskeleton of the cell. This receptor may be one of a group of plasma membrane receptors that are sensitive to the action of cytochalasins after ligand binding.

B-Lymphocytes↗
PubMed1990

Significance of transcranial Doppler CO2. Reactivity measurements for the diagnosis of hemodynamically relevant carotid obstructions.

Transcranial Doppler ultrasonography can be used to determine CO2 reactivity in the large basal cerebral arteries. CO2 reactivity is expressed as percentage increase of mean flow velocity above one volume percent of CO2, using a reference value of 40 mmHg pCO2 normalized autoregulatory reserve. A normalized autoregulatory value of 15 as the lower limit of the normal range clearly separates patients with internal carotid artery obstructions greater than or equal to 70% from the control group. Stenoses of the ipsilateral internal carotid artery of greater than or equal to 70% result in a significant decrease of normalized autoregulatory reserve in the middle cerebral artery, which can be normalized by removal of the upstream flow obstacle using a carotid thromboendarterectomy. The scatter of normalized autoregulatory values in severe internal carotid obstructions indicates the variability of collateral circulation A retrospective comparison of normalized autoregulatory reserve and ipsilateral ischemic symptoms in the supply area of the internal carotid artery reveals a significant correlation between clinical symptoms and reduced normalized autoregulatory reserve.

Adult↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Two DNA-binding proteins discriminate between the promoters of different members of the major histocompatibility complex class II multigene family.

The regulation of major histocompatibility complex (MHC) class II gene expression is a key feature of the control of normal and abnormal immune responses. In humans, class II alpha - and beta-chain genes are organized in a multigene family with three distinct subregions, HLA-DR, -DQ, and -DP. The regulation of these genes is generally coordinated, and their promoters contain highly conserved motifs, in particular the X and Y boxes. We have identified five distinct proteins that bind to specific DNA sequences within the first 145 base pairs of the HLA-DR promoter, a segment known to be functionally essential for class II gene regulation. Among these, RF-X is of special interest, since mutants affected in the regulation of MHC class II gene expression have a specific defect in RF-X binding. Unexpectedly, RF-X displays a characteristic gradient of binding affinities for the X boxes of three alpha-chain genes (DRA greater than DPA much greater than DQA). The same observation was made with recombinant RF-X. We also describe a novel factor, NF-S, which bound to the spacer region between the X and Y boxes of class II promoters. NF-S exhibited a reverse gradient of affinity compared with RF-X (DQA greater than DPA much greater than DRA). As expected, RF-X bound well to the mouse IE alpha promoter, while NF-S bound well to IA alpha. The drastic differences in the binding of RF-X and NF-S to different MHC class II promoters contrasts with the coordinate regulation of HLA-DR, -DQ, and -DP genes.

Animals↗
PubMed1989

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.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1989

Cloning of the major histocompatibility complex class II promoter binding protein affected in a hereditary defect in class II gene regulation.

The regulation of major histocompatibility complex class II gene expression is directly involved in the control of normal and abnormal immune responses. In humans, HLA-DR, -DQ, and -DP class II heterodimers are encoded by a family of alpha- and beta-chain genes clustered in the major histocompatibility complex. Their expression is developmentally controlled and normally restricted to certain cell types. This control is mediated by cis-acting sequences in class II promoters and by trans-acting regulatory factors. Several nuclear proteins bind to class II promoter sequences. In a form of hereditary immunodeficiency characterized by a defect in a trans-acting regulatory factor controlling class II gene transcription, we have observed that one of these nuclear factors (RF-X) does not bind to its target sequence (the class II X box). A cDNA encoding RF-X was isolated by screening a phage expression library with an X-box binding-site probe. The recombinant protein has the binding specificity of RF-X, including a characteristic gradient of affinity for the X boxes of HLA-DR, -DP, and -DQ promoters. RF-X mRNA is present in the regulatory mutants, indicating a defect in the synthesis of a functional form of the RF-X protein.

Base Sequence↗
PubMed1989

Inherited immunodeficiency with a defect in a major histocompatibility complex class II promoter-binding protein differs in the chromatin structure of the HLA-DRA gene.

A defect in a trans-regulatory factor which controls major histocompatibility complex class II gene expression is responsible for an inherited form of immunodeficiency with a lack of expression of human leukocyte antigen (HLA) class II antigens. We have recently described and cloned an HLA class II promoter DNA-binding protein, RF-X, present in normal B cells and absent in these class II-deficient regulatory mutants. Here we report that these in vitro results correlate with a specific change in the chromatin structure of the class II promoter: two prominent DNase I-hypersensitive sites were identified in the promoter of the HLA-DRA gene in normal B lymphocytes and found to be absent in the class II-deficient mutant cells. The same two prominent DNase I-hypersensitive sites were observed in normal fibroblastic cells induced by gamma interferon to express class II genes. Interestingly, they were also observed in the uninduced class II-negative fibroblastic cells, which have also been shown to have a normal RF-X binding pattern. We conclude that the two DNase I-hypersensitive sites in the HLA-DRA promoter reflect features in chromatin structure which correlate with the binding of the trans-acting factor RF-X and which are necessary but not sufficient for the expression of class II genes.

B-Lymphocytes↗
PubMed1988

Congenital immunodeficiency with a regulatory defect in MHC class II gene expression lacks a specific HLA-DR promoter binding protein, RF-X.

The expression of MHC class II genes is tightly regulated. One form of congenital severe combined immunodeficiency (SCID) is characterized by a regulatory defect that precludes expression of HLA class II genes. B lymphocyte cell lines from such SCID patients provide a tool for identifying putative regulatory proteins that bind to class II gene promoters. We have identified three proteins binding to specific segments of the HLA-DRA promoter, two of which interact to form the predominant DNA-protein complex observed. One of these proteins, defined as an X box binding protein (RF-X), is specifically missing in cells from class II deficient SCID patients. We propose that the molecular defect in this congenital HLA class II regulatory deficiency is a lack of RF-X and that this factor plays an important role in the normal regulation of MHC class II gene expression.

B-Lymphocytes↗
PubMed1984

Identification of Xenopus laevis mRNAs with homology to repetitive sequences.

Hybrid selection translation experiments have been carried out with genomic and cDNA relatives of two repetitive sequence families. On the basis of the in vitro translation products detected, it was found that transcripts complementary to these repeats are linked to several different mature mRNAs in stage 40 embryos of Xenopus laevis. One repeat hybridizes to mRNAs that direct the synthesis of 17 proteins. The second is present on mRNAs coding for 3 proteins. By estimating the abundance of these proteins among the translation products of total embryonic mRNA, it is inferred that all of the repeat bearing mRNAs are rare, less than one in 20,000 mRNA molecules.

Animals↗
PubMed1983

Structural analysis of repetitive sequence elements transcribed in early development of Xenopus laevis.

A cDNA library prepared from mRNA of Xenopus laevis embryos was screened with a genomic DNA fragment containing various transcribed repetitive sequence elements. Comparison of the nucleotide sequence of two isolated cDNAs and their genomic relatives allows one to define two transcribed repetitive sequence elements. One of them belongs to a highly reiterated family and consists of a tandem array of homologous subunits of 77-80 bp. The other is reiterated approximately 2200 times, has a size of 260 bp and displays a conserved region of 135 bp. The data are consistent with the presence of repetitive sequence transcripts in the 3' part of mRNA molecules.

Animals↗