Search PubMed⌕ Search

Biomedical subjects

H Clevers

Publications and source records attributed to H Clevers.

107 records · Page 6Linked to original sources
PubMed1992

The human T cell transcription factor-1 gene. Structure, localization, and promoter characterization.

We have recently isolated cDNA clones representing four alternative splice forms of a T cell-specific transcription factor, TCF-1. Here we report the characterization of the human gene encoding this factor. The TCF-1 gene is contained in 10 exons including an untranslated first exon. The DNA-binding high mobility group (HMG) box of TCF-1 is encoded by the closely spaced exons VI and VII. Differential splicing involves an alternative exon (IX) and three splice acceptor sites in exon X. Based on comparison of sequence and on the placement of an alternative exon, TCF-1 appears closely related to the recently characterized HMG box transcription factor TCF-1 alpha/LEF. In particular, the HMG boxes encoded by the two TCF genes are virtually identical. The TCF-1 gene resides on chromosome 5 band q31.1. The TCF-1 promoter coincides with a CpG island. As determined by chloramphenicol acetyltransferase analysis, the promoter is preferentially active in T cells. The promoter does not contain TCF-1/TCF-1 alpha binding sites and is therefore not autoregulated. This observation implies the existence of yet uncharacterized T cell transcription factors that are active during early T cell differentiation.

Amino Acid Sequence↗
PubMed1992

ME491 melanoma-associated glycoprotein family: antigenic identity of ME491, NKI/C-3, neuroglandular antigen (NGA), and CD63 proteins.

BACKGROUND: Numerous monoclonal antibodies (MAbs) have been produced to antigens found in human melanomas. Three of the best characterized melanoma antigens include the melanoma-associated glycoproteins (MAGs) defined by two reagent families--the ME491 family (including ME491, 8-1H, and 8-2A) and the NKI/C-3 family (including NKI/C-3 and NKI/black-13)--as well as the neuroglandular antigen (NGA) defined by MAbs LS59, LS62, and LS140. These three antigens have significant similarities in tissue distribution, biosynthesis, and structure. The ME491 MAG has been cloned, mapped, and sequenced. Numerous non-melanoma-associated proteins (Sm23, CO-029, R2, TAPA-1, CD9, CD37, CD53, and CD63) have recently been shown to have significant homology to this sequence. PURPOSE: We conducted this study to investigate the similarity between the two MAG antigens and NGA. METHODS: Several reagents defining the three different melanoma antigens were compared, using competition immunoprecipitation, immunoassay, and inhibition radioimmunoassay techniques. RESULTS: Immunoassay experiments show that MAbs defining the three melanoma antigens bind to affinity-purified ME491 antigen and inhibit each other from binding in an inhibition radioimmunoassay. Competition immunoprecipitation experiments demonstrate that the ME491 and NKI/C-3 antibodies bind to NGA. Rabbit anti-ME491 idiotype serum recognizes determinants shared by NKI/C-3 and the anti-NGA MAbs. A competition immunoprecipitation experiment also confirms the identity of CD63, as defined by MAb RUU-SP 2.28, with the three melanoma antigens. CONCLUSION: These data indicate that the MAGs defined by ME491 and NKI/C-3 as well as the anti-NGA antibodies are epitopes of the same molecule, which is identical to CD63 by both immunochemical and molecular genetic investigations. IMPLICATIONS: Our results indicate that the data obtained in studies of these three melanoma antigens may be pooled, and we propose that the molecule recognized by these reagents be classified as CD63.

Amino Acid Sequence↗
PubMed1992

Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix.

The high mobility group I (HMG) box is proposed to mediate DNA binding in a novel group of transcription-regulating proteins. Two of these, the proteins encoded by the T cell-specific TCF-1 and the mammalian sex-determining gene SRY, carry a single HMG box with specificity for the heptamer motif A/T A/T C A A A G. We have now analysed the mode of interaction of the HMG boxes of TCF-1 and SRY with this motif. Methylation interference footprinting revealed that both HMG boxes contacted adenines on both strands in the minor groove, whereas no major groove guanine contacts were discerned. Diethylpyrocarbonate (DEPC) carbethoxylation interference footprinting of TCF-1 indicated the absence of major groove contacts on positions 5, 6 and 7 of the motif. Carbethoxylation interference was observed, however, on positions 2, 3 and 4 and to a lesser extent on position 1 in the major groove. Combined T----C and A----I substitution, which changes the surface of the major groove but leaves the minor groove intact, did not interfere with sequence-specific binding by TCF-1 and SRY. These observations indicate that recognition of the heptamer motif by the HMG boxes of the distantly related TCF-1 and SRY proteins predominantly occurs through nucleotide contacts in the minor groove.

Alkylation↗
PubMed1992

Expression of GATA-3 during lymphocyte differentiation and mouse embryogenesis.

The GATA family of C4 zinc-finger transcription factors has been implicated in tissue-specific gene regulation in birds and mammals. One of the members of this family, GATA-3, is reportedly expressed specifically in the T-cell lineage, where it interacts with GATA motifs in the TCR-alpha, TCR-beta, and TCR-delta enhancers, thereby controlling the T-cell phenotype. To evaluate the differentiation control properties of GATA-3, we have now documented its expression pattern during lymphoid differentiation and murine embryogenesis. The onset of GATA-3 expression in the lymphoid lineage was studied in a panel of lymphoid (precursor) cell lines by Northern blot analysis. GATA-3 was uniquely expressed in T-lineage lymphocytes expressing TCR and CD3 genes; it was absent from TCR/CD3 mRNA-negative prothymocytes and from all B-lineage cells. In order to obtain information on the expression of GATA-3 outside the immune system, in situ hybridization was performed on mouse embryos on day 11.5-14.5 of gestation. GATA-3 mRNA was detected in fetal thymus and in erythroid cells. Outside the haemopoietic system, we detected GATA-3 mRNA throughout the central nervous system, in kidney, in the epidermis, lens fibers, the inner ear, whisker follicles, and in the primary palate. These data provide new clues about the potential role of GATA-3 during mouse development, and will aid the interpretation of currently ongoing gene knockout experiments.

Adrenal Glands↗
PubMed1991

Cloning of murine TCF-1, a T cell-specific transcription factor interacting with functional motifs in the CD3-epsilon and T cell receptor alpha enhancers.

CD3-epsilon gene expression is confined to the T cell lineage. We have recently identified and cloned a human transcription factor, TCF-1, that binds to a functional element in the T lymphocyte-specific enhancer of CD3-epsilon. In a panel of human cell lines, TCF-1 expression was restricted to T lineage cells. TCF-1 belonged to a novel family of genes that contain the so-called high mobility group 1 (HMG) box. Here we report the cloning of murine TCF-1. Two splice alternatives were identified that were not previously observed in human TCF-1. Murine and human TCF-1 displayed a 95.5% overall amino acid homology. Recombinant murine and human TCF-1 recognized the same sequence motif in the CD3-epsilon enhancer as judged by gel retardation and methylation interference assays. With the murine cDNA clones several aspects of TCF-1 were analyzed. First, deletion analysis revealed that a region of TCF-1 containing the HMG box was sufficient for sequence-specific binding. Second, by high stringency Northern blotting and in situ hybridization, TCF-1 expression was shown to be confined to the thymus and to the T cell areas of the spleen. Third, TCF-1 bound specifically to a functional T cell-specific element in the T cell receptor alpha (TCR-alpha) enhancer. The T lineage-specific expression and the affinity for functional motifs in the TCR-alpha and CD3-epsilon enhancers imply an important role for TCF-1 in the establishment of the mature T cell phenotype.

Amino Acid Sequence↗
PubMed1991

Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box.

CD3-epsilon expression is controlled by a downstream T lymphocyte-specific enhancer element. We report the identification of a T cell-specific transcription factor, TCF-1, binding to this element. The multimerized recognition motif of TCF-1 constituted a T cell-specific enhancer. Subsequent cloning of TCF-1 identified three splice alternatives. TCF-1 contained a single DNA-binding HMG box most closely related to similar boxes in the putative mammalian sex-determining gene SRY and in the Schizosaccharomyces pombe Mc mating type gene. TCF-1 mRNA was expressed uniquely in T lymphocytes. Upon cotransfection into non-T cells, TCF-1 could transactivate through its cognate motif. These results identify TCF-1 as a T cell-specific transcription factor, which might play a role in the establishment of the mature T cell phenotype.

Amino Acid Sequence↗
PubMed1991

Identification of expressed bovine class I MHC genes at two loci and demonstration of physical linkage.

A cDNA library prepared from lymphocytes of a cow (E98), homozygous at major histocompatibility complex (MHC) loci (BoLA phenotype w10, KN104), was screened with a bovine MHC class I probe. Of the cDNA clones isolated, two, (2.1 and 5.1) were selected and showed divergence at both 5' and 3' termini. E98 DNA was digested with rare-cutter enzymes (Sfi I, Mlu I, Not I, and Cla I) and fragments were size-separated by field inversion gel electrophoresis (FIGE). Hybridization with an entire class I cDNA probe revealed multiple fragments generated by each enzyme. When the 3' untranslated regions (UT) of 2.1 and 5.1 were used as probes, only one fragment was revealed in each digested sample, showing locus specificity of these probes in cattle. Further, DNA of transfected mouse fibroblasts L4 (expressing KN104) and L10 (expressing w10) hybridized to the 3'UT regions of clones 2.1 and 5.1, respectively. Northern blot analysis of the mRNA of the L4 and L10 transfected cells provided further evidence that the cDNA clones 2.1 and 5.1 code for the BoLA-KN104 and BoLA-w10 class I molecules respectively, and thus these represent the products of two different genes. A long range physical mapping of the BoLA-w10 and KN104 genes was performed using FIGE analysis of DNA of an homozygous and an heterozygous animal. This analysis revealed that the BoLA-w10 and KN104 genes are separated by not more than 210 kilobases (kb) and that they are components of a multigene family spanning 1550 kb. As the w10 gene is at the BoLA-A locus we assign the KN104 gene to a B locus.

Animals↗
PubMed1990

Identification of a bovine surface antigen uniquely expressed on CD4-CD8- T cell receptor gamma/delta+ T lymphocytes.

In this study, two monoclonal antibodies, IL-A29 and CC15, are described that identify a novel bovine cell surface marker of 215/300 kDa. The antibodies reacted with a discrete population of resting lymphocytes in peripheral blood which, in young animals, constituted about 25% of the mononuclear cells. Thymus, lymph nodes and spleen contained less than 5% positive cells. These cells were negative for surface Ig, a monocyte/granulocyte marker, and the T lymphocyte antigens CD2, CD6, CD4 and CD8. Immunohistological analyses revealed the presence of IL-A29/CC15-positive lymphocytes in the thymic medulla, in the outer cortex of lymph nodes, in the marginal zones of the spleen, in the dermal and epidermal layers of the skin and in the lamina propria of the gut. The IL-A29/CC15+ cells in unfractionated blood mononuclear cells responded in autologous and allogeneic mixed lymphocyte cultures, and when purified they responded to concanavalin A in the presence of recombinant interleukin 2. These observations suggested this population of cells belonged to the T cell lineage. In order to unambiguously define their lineage, cDNA clones encoding bovine T cell receptor (TcR) and CD3 proteins were isolated. Northern blot analyses of IL-A29/CC15+ cell populations and of established cell lines of various lineages demonstrated that they expressed TcR delta and CD3 gamma, delta and epsilon mRNA: TcR alpha was not expressed, whereas only a truncated form of TcR beta mRNA was present. These results indicate that the IL-A29 and CC15 antibodies define a unique population of CD4-CD8-, gamma/delta T cells.

Animals↗
PubMed1990

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed.

Animals↗
PubMed1989

An enhancer located in a CpG-island 3' to the TCR/CD3-epsilon gene confers T lymphocyte-specificity to its promoter.

The gene encoding the CD3-epsilon chain of the T cell receptor (TCR/CD3) complex is uniquely transcribed in all T lymphocyte lineage cells. The human CD3-epsilon gene, when introduced into the mouse germ line, was expressed in correct tissue-specific fashion. The gene was then screened for T lymphocyte-specific cis-acting elements in transient chloramphenicol transferase assays. The promoter (-228 to +100) functioned irrespective of cell type. A 1225 bp enhancer with strict T cell-specificity was found in a DNase I hypersensitive site downstream of the last exon, 12 kb from the promoter. This site was present in T cells only. The CD3-epsilon enhancer did not display sequence similarity with the T cell-specific enhancer of CD3-delta, a related gene co-regulated with CD3-epsilon during intrathymic differentiation. The CD3-epsilon enhancer was unusual in that it constituted a CpG island, and was hypomethylated independent of tissue type. Two HTLV I-transformed T cell lines were identified in which the CD3-epsilon gene was not expressed, and in which the enhancer was inactive.

Animals↗
PubMed1988

The transmembrane orientation of the epsilon chain of the TcR/CD3 complex.

The antigen receptor of the T lymphocytes is one of the most complex eukaryotic membrane structures studied to date. The T cell receptor (TcR) consists of two disulfide-linked glycoprotein chains (alpha/beta or gamma/delta) and is noncovalently associated with a group of small and invariable CD 3 proteins. Four CD 3 chains have been recognized: two highly homologous glycoproteins CD 3 gamma and delta, the more distantly related nonglycosylated CD 3 epsilon chain, and the nonglycosylated CD 3 zeta, the latter being present as a homodimer. The unraveling of the architecture of the TcR/CD 3 complex is crucial to our understanding of the processes underlying its assembly, recognition and transmembrane signaling. The transmembrane orientation of the TcR chains and of CD 3 gamma and CD 3 delta can be directly inferred from their primary structure, based on the presence of concensus N-linked glycosylation sites N-terminal of their transmembrane domains. This prediction can not be made, however, for nonglycosylated molecules like the CD 3 epsilon chain. In order to determine the transmembrane orientation of CD 3 epsilon, anti-peptide antisera directed against the N-termini of the human and murine CD 3 epsilon chains were generated in rabbits. Both antisera stained intact T cells, demonstrating that the N-terminus of the CD 3 epsilon chain was located at the outer surface of the plasma membrane. The anti-human CD 3 epsilon peptide antiserum was found to be mitogenic for peripheral blood T cells, a finding previously reported only for monoclonal anti-TcR/CD 3 reagents. Using a novel transient expression system in murine T lymphocytes, the human CD 3 epsilon chain could be expressed on the surface of CD 3+, but not CD 3- murine T cells, as indicated by fluorescence staining with the anti-peptide antiserum. This experiment confirmed the specificity of the anti-peptide antiserum and, perhaps more importantly, indicated that the human CD 3 epsilon chain was correctly assembled in the murine CD 3 complex. Moreover, the anti-human CD 3 monoclonal antibody UCHT1 was found to stain T cells expressing the human CD 3 epsilon chain.

Amino Acid Sequence↗
PubMed1988

Characterization and expression of the murine CD3-epsilon gene.

The receptor for antigen on the surface of T lymphocytes consists of a variable disulfide-bridged hetero-dimer (TCR-alpha/beta or -gamma/delta) associated with invariant CD3 proteins (CD3-gamma, -delta, -epsilon, and -zeta). The genes coding for the CD3 proteins are expressed in the earliest recognizable thymocytes, preceding the rearrangement and expression of the TCR genes. The isolation, characterization, and in vitro expression of the murine CD3-epsilon gene, as reported here, represent obligatory steps toward our understanding of the complex rules that govern T-cell-specific gene expression. The CD3-epsilon gene was transcribed from a non-TATA promoter and consisted of eight exons, two of which were unusually small (18 and 15 base pairs). The transmembrane exon was found to be homologous to the transmembrane exons of the CD3-gamma and CD3-delta genes. In transient-transfection experiments, a genomic fragment comprising 4 kilobases of upstream sequence and extending into the second exon sufficient to drive the expression of a reporter gene in murine T cells.

Amino Acid Sequence↗
PubMed1987

Evolutionary relationship between the T3 chains of the T-cell receptor complex and the immunoglobulin supergene family.

Antigen receptors on the surface of the thymus-derived (T) lymphocytes are associated with small integral membrane proteins called the T3 (CD3) gamma, delta, epsilon, and zeta chains. After interaction of the T-cell receptor with antigen, the T3 proteins are believed to transfer an activation signal to the intracellular compartment. In previous studies, the human gamma, epsilon, and delta chains have been cloned along with the mouse delta chain, but a relationship between these sequences and known molecular families has not been established. We now report the molecular cloning and characterization of the murine T3-epsilon protein and a sequence and structural analysis of the relationships between all the T3 chains and the immunoglobulin superfamily. It is established that the T3 chains are immunoglobulin-related and a particular relationship to the neural cell adhesion molecule (N-CAM) is noted. This sequence relationship adds interest to previous findings that the T3 chains are genetically linked to N-CAM and Thy-1 antigen on band q23 of human chromosome 11.

Animals↗
PubMed1987

Close linkage of the mouse and human CD3 gamma- and delta-chain genes suggests that their transcription is controlled by common regulatory elements.

Antigen receptors on the T-cell surface are noncovalently associated with at least four invariant polypeptide chains, CD3-gamma, -delta, -epsilon, and -zeta. The mouse CD3-gamma gene, consisting of seven exons, was found to be highly homologous to the CD3-delta gene described earlier. Both the high level of sequence homology and the exon/intron organization indicate that the CD3-gamma and -delta genes arose by gene duplication. Surprisingly, murine and human genomic DNA clones could be isolated that contained elements of both the CD3-gamma and CD3-delta genes. In fact, the putative transcription start site of the mouse CD3-gamma gene is less than 1.4 kilobases from the transcription initiation site of the mouse CD3-delta gene. Common elements that regulate the divergent transcription of the two genes are therefore proposed to be located in the intervening 1.4-kilobase DNA segment. This might contribute to the coordinate expression of the CD3-gamma and -delta genes during intrathymic maturation of T lymphocytes.

Amino Acid Sequence↗