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C Ucla

Publications and source records attributed to C Ucla.

17 recordsLinked to original sources
PubMed2004

The transcription factor RFX3 directs nodal cilium development and left-right asymmetry specification.

There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion, RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.

Animals↗
PubMed2001

From PREDs and open reading frames to cDNA isolation: revisiting the human chromosome 21 transcription map.

A supernumerary copy of human chromosome 21 (HC21) causes Down syndrome. To understand the molecular pathogenesis of Down syndrome, it is necessary to identify all HC21 genes. The first annotation of the sequence of 21q confirmed 127 genes, and predicted an additional 98 previously unknown "anonymous" genes (predictions (PREDs) and open reading frames (C21orfs)), which were foreseen by exon prediction programs and/or spliced expressed sequence tags. These putative gene models still need to be confirmed as bona fide transcripts. Here we report the characterization and expression pattern of the putative transcripts C21orf7, C21orf11, C21orf15, C21orf18, C21orf19, C21orf22, C21orf42, C21orf50, C21orf51, C21orf57, and C21orf58, the GC-rich sequence DNA-binding factor candidate GCFC (also known as C21orf66), PRED12, PRED31, PRED34, PRED44, PRED54, and PRED56. Our analysis showed that most of the C21orfs originally defined by matching spliced expressed sequence tags were correctly predicted, whereas many of the PREDs, defined solely by computer prediction, do not correspond to genuine genes. Four of the six PREDs were incorrectly predicted: PRED44 and C21orf11 are portions of the same transcript, PRED31 is a pseudogene, and PRED54 and PRED56 were wrongly predicted. In contrast, PRED12 (now called C21orf68) and PRED34 (C21orf63) are now confirmed transcripts. We identified three new genes, C21orf67, C21orf69, and C21orf70, not previously predicted by any programs. This revision of the HC21 transcriptome has consequences for the entire genome regarding the quality of previous annotations and the total number of transcripts. It also provides new candidates for genes involved in Down syndrome and other genetic disorders that map to HC21.

Animals↗
PubMed2001

Interferon-alpha-induced endogenous superantigen. a model linking environment and autoimmunity.

We earlier proposed that a human endogenous retroviral (HERV) superantigen (SAg) IDDMK(1,2)22 may cause type I diabetes by activating autoreactive T cells. Viral infections and induction of interferon-alpha (IFN-alpha) are tightly associated with the onset of autoimmunity. Here we establish a link between viral infections and IFN-alpha-regulated SAg expression of the polymorphic and defective HERV-K18 provirus. HERV-K18 has three alleles, IDDMK(1,2)22 and two full-length envelope genes, that all encode SAgs. Expression of HERV-K18 SAgs is inducible by IFN-alpha and this is sufficient to stimulate V beta 7 T cells to levels comparable to transfectants constitutively expressing HERV-K18 SAgs. Endogenous SAgs induced via IFN-alpha by viral infections is a novel mechanism through which environmental factors may cause disease in genetically susceptible individuals.

Antigens, CD↗
PubMed1997

Two novel mutations in the MHC class II transactivator CIITA in a second patient from MHC class II deficiency complementation group A.

Congenital MHC class II deficiency or bare lymphocyte syndrome (BLS; McKusick 209920) is caused by defects in trans-acting regulatory factors that control MHC class II expression and is therefore a disease of gene regulation. There are at least four complementation groups and the genetic and molecular dissection of this rare disease has contributed considerably to our current understanding of the molecular mechanisms governing MHC class II expression. Identification of the gene that is defective in BLS complementation group A, CIITA (MHC class II transactivator), has led to the discovery that CIITA acts as a master control factor of MHC class II expression. We have identified the CIITA mutations in a second patient from BLS group A. Two novel mutations abolish CIITA function, as shown by transfection experiments. Molecular analysis of these two novel mutations, together with the one described earlier in the first patient, is informative in terms of CIITA structure-function relationships.

Alleles↗
PubMed1997

Efficient repression of endogenous major histocompatibility complex class II expression through dominant negative CIITA mutants isolated by a functional selection strategy.

Major histocompatibility complex class II (MHC-II) molecules present peptide antigens to CD4-positive T cells and are of critical importance for the immune response. The MHC-II transactivator CIITA is essential for all aspects of MHC-II gene expression examined so far and thus constitutes a master regulator of MHC-II expression. In this study, we generated and analyzed mutant CIITA molecules which are able to suppress endogenous MHC-II expression in a dominant negative manner for both constitutive and inducible MHC-II expression. Dominant negative CIITA mutants were generated via specific restriction sites and by functional selection from a library of random N-terminal CIITA deletions. This functional selection strategy was very effective, leading to strong dominant negative CIITA mutants in which the N-terminal acidic and proline/serine/threonine-rich regions were completely deleted. Dominant negative activity is dependent on an intact C terminus. Efficient repression of endogenous MHC-II mRNA levels was quantified by RNase protection analysis. The quantitative effects of various dominant negative CIITA mutants on mRNA expression levels of the different MHC-II isotypes are very similar. The optimized dominant negative CIITA mutants isolated by functional selection should be useful for in vivo repression of MHC-II expression.

Burkitt Lymphoma↗
PubMed1996

Locations of human and mouse genes encoding the RFX1 and RFX2 transcription factor proteins.

RFX transcription factors constitute a highly conserved family of site-specific DNA binding proteins involved in the expression of a variety of cellular and viral genes, including major histocompatibility complex class II genes and genes in human hepatitis B virus. Five members of the RFX gene family have been isolated from human and mouse, and all share a highly characteristic DNA binding domain that is distinct from other known DNA binding motifs. The human RFX1 and RFX2 genes have been assigned by in situ hybridization to chromosome 19p13.1 and 19p13.3, respectively. In this paper, we present data that localize RFX1 and RFX2 precisely within the detailed physical map of human chromosome 19 and genetic data that assign Rfx1 and Rfx2 to homologous regions of mouse chromosomes 8 and 17, respectively. These data define the established relationships between these homologous mouse and human regions in further detail and provide new tools for linking cloned genes to phenotypes in both species.

Animals↗
PubMed1994

RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins.

RFX1 is a transactivator of human hepatitis B virus enhancer I. We show here that RFX1 belongs to a previously unidentified family of DNA-binding proteins of which we have cloned three members, RFX1, RFX2, and RFX3, from humans and mice. Members of the RFX family constitute the nuclear complexes that have been referred to previously as enhancer factor C, EP, methylation-dependent DNA-binding protein, or rpL30 alpha. RFX proteins share five strongly conserved regions which include the two domains required for DNA binding and dimerization. They have very similar DNA-binding specificities and heterodimerize both in vitro and in vivo. mRNA levels for all three genes, particularly RFX2, are elevated in testis. In other cell lines and tissues, RFX mRNA levels are variable, particularly for RFX2 and RFX3. RFX proteins share several novel features, including new DNA-binding and dimerization motifs and a peculiar dependence on methylated CpG dinucleotides at certain sites.

Alternative Splicing↗
PubMed1993

Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2.

Defects in human DNA repair proteins can give rise to the autosomal recessive disorders xeroderma pigmentosum (XP) and Cockayne's syndrome (CS), sometimes even together. Seven XP and three CS complementation groups have been identified that are thought to be due to mutations in genes from the nucleotide excision repair pathway. Here we isolate frog and human complementary DNAs that encode proteins resembling RAD2, a protein involved in this pathway in yeast. Alignment of these three polypeptides, together with two other RAD2 related proteins, reveals that their conserved sequences are largely confined to two regions. Expression of the human cDNA in vivo restores to normal the sensitivity to ultraviolet light and unscheduled DNA synthesis of lymphoblastoid cells from XP group G, but not CS group A. The XP-G correcting protein XPGC is generated from a messenger RNA of approximately 4 kilobases that is present in normal amounts in the XP-G cell line.

Amino Acid Sequence↗
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↗
PubMed1990

Interferon gamma drastically modifies the regulation of interleukin 1 genes by endotoxin in U937 cells.

IL-1 alpha, IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) gene expression is induced by LPS (endotoxin) in monocytes/macrophages and in some monocytic cell lines. IFN gamma and 1 alpha,25-dihydroxyvitamin D3 (1,25[OH]2D3) are important macrophage-activating factors. They induce changes in the human monocyte cell line U937 that reflect cellular differentiation. We have studied the effect of IFN-gamma and of 1,25(OH)2D3 on the expression of IL-1 and TNF-alpha messenger RNA in response to LPS. The induction of these genes by LPS is immediate and transient, with a maximum in 3 h. Preincubation of the cells with IFN-gamma or with 1,25(OH)2D3 increases these mRNA responses to LPS about fourfold. More importantly, cells exposed to IFN-gamma for 72 h exhibit a drastically different and unexpected pattern of IL-1 alpha and IL-1 beta gene response to LPS. Instead of the normal transient response, one then observes a sustained increase in IL-1 alpha and IL-1 beta gene expression over at least 16 h after LPS stimulation. This was measured both at the level of mRNA and by direct transcription assays (run-off). This striking effect of IFN-gamma on the kinetics of IL-1 gene response does not apply to the TNF-alpha gene. Interestingly, 1,25(OH)2D3, which shares with IFN-gamma a number of important effects on monocytes/macrophages, does not affect the kinetics of IL-1 gene response to LPS. In view of the biological relevance of endotoxin as a macrophage activator, the potential clinical implication of this prolonged induction of IL-1 gene expression is discussed.

Blotting, Northern↗
PubMed1989

Correlation of structure with T cell responses of the three members of the HLA-DRw52 allelic series.

A third allele at the DRB3 locus, DRw52c, represents an intermediate sequence between DRw52a and DRw52b and may have arisen by a gene conversion-like event. The recognition of cells bearing these molecules by a number of alloreactive and antigen-specific DR-restricted T cell clones was analyzed. On the basis of a theoretical model of HLA class II structure, distinct amino acid clusters have been identified as motifs controlling TCR recognition. These are located both in the cleft and in the alpha-helical edge of the MHC class II recognition platform. Motifs shared between two alleles may restrict public T cell clones.

Alleles↗
PubMed1989

Differential expression and regulation of the glucokinase gene in liver and islets of Langerhans.

Glucokinase, a key regulatory enzyme of glucose metabolism in mammals, provides an interesting model of tissue-specific gene expression. The single-copy gene is expressed principally in liver, where it gives rise to a 2.4-kilobase mRNA. The islets of Langerhans of the pancreas also contain glucokinase. Using a cDNA complementary to rat liver glucokinase mRNA, we show that normal pancreatic islets and tumoral islet cells contain a glucokinase mRNA species approximately 400 nucleotides longer than hepatic mRNA. Hybridization with synthetic oligonucleotides and primer-extension analysis show that the liver and islet glucokinase mRNAs differ in the 5' region. Glucokinase mRNA is absent from the livers of fasted rats and is strongly induced within hours by an oral glucose load. In contrast, islet glucokinase mRNA is expressed at a constant level during the fasting-refeeding cycle. The level of glucokinase protein in islets measured by immunoblotting is unaffected by fasting and refeeding, whereas a 3-fold increase in the amount of enzyme occurs in liver during the transition from fasting to refeeding. From these data, we conclude (i) that alternative splicing and/or the use of distinct tissue-specific promoters generate structurally distinct mRNA species in liver and islets of Langerhans and (ii) that tissue-specific transcription mechanisms result in inducible expression of the glucokinase gene in liver but not in islets during the fasting-refeeding transition.

Animals↗
PubMed1987

Molecular cloning of glucokinase cDNA. Developmental and dietary regulation of glucokinase mRNA in rat liver.

A rat liver cDNA library enriched for glucokinase sequences was constructed using the phage expression vector lambda gt11 and screened with an antiserum to glucokinase. A positive phage clone termed lambda-GK223 was isolated by several rounds of plaque purification. When introduced in the high frequency lysogenization strain Y1089, the phage was shown to encode a fusion protein containing epitopes specific to rat liver glucokinase. The 1800-base pair cDNA insert of lambda-GK223 was subcloned in a pUC plasmid, and a resulting recombinant termed pUC-GK1 was used for hybrid selection of mRNA. The selected mRNA directed the synthesis in a cell-free translation system of a protein identified as glucokinase by electrophoresis and immunoprecipitation. The cloned cDNA was then used as a probe to measure the amount of glucokinase mRNA in rat liver during postnatal development. Glucokinase mRNA, 2.4 kilobases in length, was first detectable at day 14 after birth and increased 40-fold in amount from this age to day 31, in parallel with the emergence of glucokinase enzyme activity. In the adult rat, glucokinase mRNA was low during fasting and increased more than 50-fold above the fasting level within 6 h of an oral glucose load. However, maximal accumulation of glucokinase mRNA was short-lived and the mRNA level returned toward basal values by 18 h of refeeding. These data point to rapid and massive effects on the expression of the glucokinase gene at the transcriptional or post-transcriptional levels during ontogenic development and dietary changes in the adult animal.

Animals↗
PubMed1987

Analysis of HLA-D micropolymorphism by a simple procedure: RNA oligonucleotide hybridization.

Recent progress in the molecular genetics of HLA class II antigens has revealed the existence of multiple loci and of a large degree of polymorphism, with more individual alleles than was expected. An accurate detection and analysis of this extensive polymorphism is essential for optimal HLA typing for transplantation and for a reevaluation of HLA-disease association. Because of the limitations of the current typing methods, including restriction fragment length polymorphisms, we have proposed a DNA typing procedure based on hybridization with loci- and allele-specific oligonucleotides. Here we present a much simpler way of analyzing class II micropolymorphism down to the level of single nucleotide differences. RNA oligonucleotide typing (ROT) relies on RNA dot blots and requires 10-20 ml of blood. It is shown that with appropriate oligonucleotide probes, ROT can reliably and unambiguously identify any polymorphism at any of the HLA loci, including new alleles, not identified with previous methods. This illustrates the importance of oligonucleotide typing to optimize HLA matching, in particular for transplantation involving unrelated donors.

Alleles↗
PubMed1984

Induction of human interleukin 1 mRNA measured by collagenase- and prostaglandin E2-stimulating activity in rheumatoid synovial cells.

Human blood peripheral monocyte/macrophages release in culture a mononuclear cell factor (MCF) which stimulates the production of collagenase and prostaglandin E2 by human rheumatoid synovial cells and dermal fibroblasts. These two products play a role in connective tissue destruction. MCF has an apparent molecular weight of approximately 15 000 and is biologically and biochemically indistinguishable from interleukin 1. MCF therefore belongs to the well-documented nonimmune biological activities attributed to interleukin 1. Studies on the mechanisms of production and action of such monokine(s) have been difficult in view of the minute quantities produced by freshly isolated cells or from human monocytic lines. Starting from lectin-stimulated human blood mononuclear cells, we have isolated poly(A)+ RNA and studied its translation following microinjection into Xenopus laevis oocytes. The mRNA translation products stimulated collagenase and prostaglandin E2 production in human rheumatoid synovial cells and dermal fibroblasts. The size of MCF-mRNA was estimated to be 10 S. The mRNA of a member of the interleukin 1 family can now be studied in a system based on a specific and direct relevant biological assay and eventually compared with those of other monokines.

Animals↗