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Animals↗
Biomedical subjects
C Auffray
Publications and source records attributed to C Auffray.
Chicken tyrosine hydroxylase gene: isolation and functional characterization of the 5' flanking region.
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines. We describe here the isolation of the chicken TH gene and the analysis of 3 kb of its 5' flanking region. The chicken TH transcription unit spans 19 kb. The 60-bp proximal promoter contains a TATA box and a cyclic AMP response element (CRE) sequence. The 5' flanking region contains several AP1-, AP2-, and octamer-like sequences as well as a glucocorticoid response element at position -1.4 kb. A construct containing the 3-kb 5' flanking DNA fused to the chloramphenicol acetyltransferase (CAT) gene was transiently transfected into PC12 cells, and the effect of various effectors was tested. Only forskolin increased the CAT activity, likely owing to the presence of the CRE sequence. Constructs prepared by progressively deleting the 5' flanking DNA were transfected into PC12 and QT6 (quail transformed fibroblasts) cells. In both cell types, the transcriptional activity increased with deletion of the 5' flanking region. These results show that the 60-bp region containing the TATA box and the CRE is sufficient to act as a constitutive promoter for the chicken TH gene and that this region appears to be negatively controlled by upstream sequences.
Adrenal Glands↗
Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens.
The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3-expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial characterization will guide further studies on the functions of this molecule, which may play an important role in immune responses mediated by T and NK lymphocytes.
Amino Acid Sequence↗
Cloning and mapping of 5' exons from the gene encoding chicken beta nerve growth factor.
An NGF cDNA containing the 5' exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5' portion.
Animals↗
New chamber for flow cytometric analysis over an extended range of stream velocity and application to cell adhesion measurements.
When analyzed in a flow cytometer, particles are suddenly accelerated to high velocities (1-10 m.s-1) over very short distances. This feature is essential to obtain high analysis rates and low coincidence levels, but translates into very strong velocity gradients (greater than 10(5) s-1): particles experience strong hydrodynamic stresses that elongate them and tend to dissociate weakly associated complexes. In order to analyze fragile conjugates formed by heterotypic adhesion between two cell types, a flow cytometer was modified to make hydrodynamic stress not only much weaker but also adjustable. A new and easily adaptable flow cell was designed for the instruments of the FACS series; it provided satisfactory hydrodynamic conditions on a wide continuous range of flow rates. Accompanying electronic adaptations permitted standard analysis between 0.01 and 10 m.s-1. At 0.01 m.s-1, the velocity gradient roughly amounts to 50 s-1. Conjugates formed by the adhesion between human B and resting T lymphocytes, disrupted in conventional flow cytometers, could be detected and precisely quantified provided analysis velocity was kept below 0.1 m.s-1. We conclude that low velocity flow cytometry makes possible the quantification of weak intercellular adhesion phenomena, and is potentially useful for the future development of new biomechanical techniques and other applications.
B-Lymphocytes↗
Interferon induction of chicken MHC class I gene expression: phylogenetic conservation of the interferon-responsive element.
The 5' upstream region of a chicken MHC class I gene BF-IV contains sequence motifs similar to the interferon consensus sequences (ICS) contained in promoters of many mammalian interferon-regulated genes. To study a possible functional role of this putative chicken ICS, an oligonucleotide spanning the upstream sequences of the BF-IV gene (-174/-194) was cloned singly or in multiple copies before the herpes TK promoter controlling the chloramphenicol acetyl transferase (CAT) gene (pBLCAT2). Transient expression studies performed with primary chicken fibroblasts (CEF) showed that the chicken ICS represses constitutive promoter activity. The chicken ICS, however, enhanced CAT activity up to 20-fold following treatment with chicken interferon (IFN). Deletion analysis of the BF-IV promoter also confirms that the upstream DNA sequences (-174/-194) contain a functional ICS recognized by chicken interferon. The murine ICS of the H2-Ld gene was also activated by chicken interferon when introduced into CEF. IFN activation of chicken ICS containing reporters was also observed in transformed chicken fibroblast lines. We show that the chicken ICS binds two specific nuclear factors present in chicken fibroblasts which are induced by interferon. These factors were also capable of recognizing the mouse ICS, suggesting the conservation of a relevant DNA-binding protein. Taken together, these data indicate that the chicken ICS motif contained in a sequence from -174 to -194 of the BF-IV gene acts as a strong interferon-response element, which has been functionally conserved during about 270 million years of separate evolution of mammals and birds.
Animals↗
Mapping the beta NGF gene in situ to a microchromosome in chicken.
The chicken nerve growth factor (beta NGF) gene has been mapped by fluorescent in situ hybridization to a pair of microchromosomes, confirming previous reports of the existence of a single gene locus. A 39-kb genomic fragment cloned in a cosmid vector and including the 5' end of the beta NGF locus was biotinylated for nonradioactive detection of the gene. This report adds to the increasing evidence proving microchromosomal localization of highly conserved and biologically fundamental genes. The implications of such genes belonging to very small linkage groups for the transmission of alleles from generation to generation together with the relevance of nonisotopic in situ hybridization for avian gene mapping are considered.
Animals↗
Assignment of the chicken tyrosine hydroxylase gene to chromosome 6 by FISH.
The gene for tyrosine hydroxylase, the first and rate-limiting enzyme in the biosynthetic pathway of catecholamine neurotransmitters, has been localized in situ to chromosome 6 in the chicken. It is the first DNA marker to be reported for this telocentric macrochromosome. Use of a 45-kb biotinylated chicken-specific cosmid probe and a sensitive fluorescent detection system proved to be highly efficient, with over 90% of metaphases showing positive hybridization signals on one or (usually) both chromosome 6 homologs, in physically mapping this single-gene locus.
Animals↗
Mutations in the D strand of the human CD4 V1 domain affect CD4 interactions with the human immunodeficiency virus envelope glycoprotein gp120 and HLA class II antigens similarly.
CD4, a cell surface glycoprotein expressed primarily by T lymphocytes and monocytes, interacts with HLA class II antigens to regulate the immune response. In AIDS, CD4 is the receptor for the human immunodeficiency virus, which binds to CD4 through envelope glycoprotein gp120. Delineation of the ligand-binding sites of CD4 is necessary for the development of immunomodulators and antiviral agents. Although the gp120 binding site has been characterized in detail, much less is known about the class II binding site, and it is as yet uncertain whether they partially or fully overlap. To investigate CD4 binding sites, a cellular adhesion assay between COS cells transiently transfected with CD4 and B lymphocytes expressing HLA class II antigens has been developed that is strictly dependent on the CD4--class II interaction, quantitative, and highly reproducible. Mutants of CD4 expressing amino acids with distinct physicochemical properties at positions Arg-54, Ala-55, Asp-56, and Ser-57 in V1, the first extracellular immunoglobulin-like domain, have been generated and studied qualitatively and quantitatively for interaction with HLA class II antigens, for membrane expression, for the integrity of CD4 epitopes recognized by a panel of monoclonal antibodies, and for gp120 binding. The results obtained show that the mutations in this tetrapeptide, which forms the core of a synthetic peptide previously shown to have immunosuppressive properties, affect the two binding functions of CD4 similarly, lending support to the hypothesis that the human immunodeficiency virus mimicks HLA class II binding to CD4.
Amino Acid Sequence↗
Primary structure and ontogeny of an avian CD3 transcript.
The structure of a chicken CD3 chain has been determined by isolating a cDNA clone (T11.15) that encodes a 175-amino-acid-long protein, including the NH2-terminal signal peptide. In Northern blot experiments, the earliest expression of the T11.15 transcript was detected in the thymus at embryonic day 10 (i.e., 1 day after cytoplasmic expression of a CD3 epitope recognized by a specific monoclonal antibody [CT3; Chen, C.L.H., Ager, L.L., Gartland, G.L. & Cooper, M.D. (1986) J. Exp. Med. 164, 375-380], but 2 days before the appearance of clonotypic components of the T-cell antigen receptor). Sequence similarity of this chicken protein sequence compared with that of the known mammalian CD3 gamma and delta polypeptides was 36-39% and 39-40%, respectively. Amino acid sequence alignments between avian and mammalian CD3 revealed maximum conservation in the transmembrane and cytoplasmic domains as well as in the regions flanking the cysteine residues in the extracellular domain, underlining their functional importance. The difficulty of unambiguously assigning this chain to a single mammalian CD3 subunit on the basis of sequence comparison raises the possibility that this polypeptide represents a derivative from an ancestral form of the gamma and delta chains. It is thus possible that a single chain may play the role of both CD3 gamma and delta subunits in the chicken CD3 complex or, alternatively, that gene duplications occurred independently in the avian and mammalian lineages.
Amino Acid Sequence↗
Interaction of CD4 with HLA class II antigens and HIV gp120.
We have developed a cellular adhesion assay in which B lymphocytes expressing HLA class II antigens form rosettes with COS cells expressing high levels of cell surface CD4 upon transient transfection with a CDM8-CD4 plasmid construct. The assay is specific, quantitative, and overcomes the difficulties encountered with a previously described system using an SV40 viral vector. Rosette formation was inhibited by a series of CD4- and HLA-DR-specific antibodies, as well as by human immunodeficiency virus (HIV) gp 120, and a synthetic peptide derived from part of its binding site for CD4 (amino acid residues 414-434), but not by a variety of other effectors, including several soluble CD4 derivatives. The comparison of this pattern of inhibition with those observed in other systems further emphasizes the great similarity, but incomplete identity, in the CD4 binding sites for HLA class II antigens and HIV gp120, and supports a model in which CD4 is considered as an allosteric servomodulator of T-cell adhesion and function which probably is induced to interact with HLA class II antigens when associated with the Tcr/CD3 complex.
Allosteric Regulation↗
Evolutionary relationship between human and mouse immunoglobulin kappa light chain variable region genes.
Similar to the Igh-V multigene family, the human or mouse Igk-V repertoire is a distorted continuum of homologous genes that may be grouped into families displaying greater than 80% nucleic acid sequence similarity among their members. Systematic interspecies sequence comparisons reveal that most human Igk-V gene families exhibit clear homology to mouse Igk-V families (sequence similarity generally greater than 74%). A hypothetical phylogenetic tree of Igk-V genes predicts that a minimum of seven Igk-V genes/families predate mammalian radiation. In two cases, several interrelated mouse Igk-V families exhibit phylogenetic equidistance with just one human Igk-V family, implying a more pronounced divergence for the elevated number of Igk-V gene families in the mouse. Mouse-human Igk-V comparisons, moreover, illustrate how expansion, contraction, and perhaps deletion of Igk-V gene families shape the Igk-V repertoire during mammalian evolution.
Animals↗
The molecular biology of MHC genes.
Antigenic peptides become associated with major histocompatibility complex (MHC) class I and class II surface antigens, are then presented to T cells and thereby elicit an antigen-specific cellular or humoral immune response. MHC molecules are genetically heterogeneous and polymorphic; their structure is therefore relevant to modulation of the immune system. The selective pressure resulting from this modulation is in turn the main driving force for the evolution of the complex genetic system. The density of MHC molecules on the cell surface is another parameter that influences immune responsiveness. The study of the evolution and regulation of MHC genes is, therefore, of great interest. These and other themes were discussed at the Third IIGB Workshop* which was recently held in Capri.
Animals↗
CD4-targeted immune intervention: a strategy for the therapy of AIDS and autoimmune disease.
The cell-surface antigen CD4 plays a pivotal role in the class II MHC-restricted response of specific T lymphocytes, and serves as the major receptor of human immunodeficiency virus (HIV). The recent elucidation of CD4 function in physiological and pathological conditions has improved prospects for CD4-targeted immune therapy by facilitating the design of therapeutic strategies aimed at blocking CD4 function, delivering immunosuppressive signals via this receptor molecule, or selectively depleting CD4+ cells.
Acquired Immunodeficiency Syndrome↗
Down regulation of T-cell adhesion by CD4.
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Antigens, Differentiation, T-Lymphocyte↗
Regulation of T helper-B lymphocyte adhesion through CD4-HLA class II interaction.
Antigen-independent adhesion of CD4+ T lymphocytes to Epstein-Barr virus (EBV)-transformed B cells is mediated by CD2/lymphocyte function-associated antigen (LFA)-3 and LFA-1/intracellular adhesion molecule (ICAM)-1. Although some anti-CD4 antibodies block the antigen-independent adhesion of CD4+ T lymphocytes, the CD4-HLA class II interaction does not appear to significantly contribute to the forces of cell adhesion since CD4+ T cells equally bind HLA class II+ and HLA class II- mutant B cells. In addition, conjugates formed between CD4+ T cells and HLA class II- B cells remain stable for at least 1 h while CD4+T/HLA class II+ B cell conjugate percentages promptly drop off. Down-regulation of CD4 or spontaneous low expression of CD4 also results in a persistance of conjugates formed with B cells. The role of the CD4-HLA class II interaction has been further studied by investigating the inhibitory effect of synthetic 12-mer peptides analogous to HLA class II and containing the Arg-Phe-Asp-Ser sequence conserved in the beta 1 domain. These peptides were previously found to inhibit HLA class II-restricted T cell responses, this sequence being thought to be involved in CD4-HLA class II interaction. These peptides block conjugate formation of CD4+ resting T cells or clones but not of CD8+ T cells, by interacting with the T cells as shown by preincubation experiments. Down-regulation of CD4 or spontaneous low expression results in the loss of the inhibitory activity. The peptide-mediated inhibition is neutralized by a soluble dimeric CD4 molecule. Alteration within the Arg-Phe-Asp-Ser sequence results in a significant loss of inhibition. It is thus proposed that the CD4-HLA class II interaction negatively regulates antigen-independent adhesion of T cells, this interaction involving the highly conserved Arg-Phe-Asp-Ser sequence in the HLA class II beta 1 sequence as a CD4-binding site.
Amino Acid Sequence↗
Immunohistochemical localisation of nerve growth factor in a subpopulation of chick spinal ganglion neurons.
We have previously isolated and sequenced the chicken nerve growth factor (NGF) gene and now, from the deduced amino acid sequence, selected and produced peptides suitable for use as antigens. Anti-sera raised against these peptides inhibit the biological activity of a partially purified preparation of native chicken NGF and, when used in immunocytochemical studies, allow the visualisation of sensory neurons accumulating endogenous NGF. Immunoreactive cells form a distinct population of small neurons which may correspond to the well-described neurons generated in the dorsomedial area of spinal ganglia. We conclude that two subpopulations of neurons exist within dorsal root ganglia, whose requirements for, and use of, NGF may be quite distinct.
Amino Acid Sequence↗