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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The c-myb oncogene encodes a nuclear binding protein which may play a major role in differentiation during early T cell development. However, the functionally important transcription regions in the GC promoter site have not been defined and the significance of the regulation of this promoter site in T cell differentiation has not been determined. Therefore, the promoter strength was determined by measurement of the CAT activity in cell extracts of EL-4 cells that were transfected with a CAT expression vector that contained cloned segments of the 5' myb gene. Stepwise removal of DNA sequences between -2300 bp and -346 bp upstream from the ATG initiation codon resulted in a gradual loss of 50% of CAT activity, whereas deletion of DNA sequences from -346 to -295 and -232 to -155 bp upstream from the ATG initiation codon eliminated promoter activity. On analysis of the CAT activity after transfection of various cell lines with these same constructs, it was found that the same two promoter regions were required for high CAT activity in all the cell lines, including murine cell lines which express the alpha/beta TCR and high levels of c-myb (BW5147), the alpha/beta TCR and low levels of c-myb (Yac-1), or the gamma/delta TCR (KN 12.1 and KN 2.4 T), a murine fibroblast T cell line (NIH-3T3), and a human epithelial cell line (HeLa). However, the CAT activity did not correlate with steady state levels of expression of the c-myb gene in the murine cell lines. Our data indicate that the c-myb oncogene promoter is constitutively expressed is highly dependent on a limited region of the 5' myb gene, requires two DNA elements for optimal activity, and is functional in diverse T cell lines.
Mol Immunol 1993 Jun
PMID:Regulation of c-myb oncogene expression in immature and mature murine T cells. 832 Dec 45

We have designed a convenient procedure for the analysis of V beta repertoire expression in polyclonal T-cell populations. In this procedure T-cell RNA is converted to cDNA, polydC-tailed with terminal deoxynucleotidyl transferase and submitted to one-side specificity PCR amplification with a constant region oligonucleotide primer. The amplified material is then analysed by reverse spot-test hybridization: after 32P-labelling, the amplification product is put to hybridize on a membrane where specially designed V beta subfamily-specific probes are immobilized. The radioactivity fixed on each probe can then be easily quantified and the signal obtained is directly proportional to the initial amount of homologous RNA. We applied this technique to the study of V beta gene selection following T-cell stimulation by staphylococcal enterotoxins B and E. We show that with these toxins two almost non-overlapping sets of T-cells are recruited and that this selection is likely to be dependent on specific amino acid residues shaping the fourth complementarity determining region of the TCR-beta chain. These residues constitute two tandemly-conserved tripeptide sequences (Asp39Pro40Gly41)-(Val69Ser70Arg71) and (Arg66Phe67Ser68)-(Asp88Ser89Ala90) in the SEB- and the SEE-responsive V beta gene clusters respectively.
Mol Immunol 1993 Jul
PMID:An alternative method for T-cell receptor repertoire analysis: clustering of human V-beta subfamilies selected in responses to staphylococcal enterotoxins B and E. 834 Dec 82

The recombination activating genes RAG-1 and RAG-2 appear to be necessary components of the machinery needed for the Ig or TCR gene rearrangements that occur in developing B and T lymphocytes. In addition RAG-2 has been implicated in the process of V-gene diversification by somatic gene conversion in the chicken. Because gene conversion may be an important mechanism for V-gene diversification in the rabbit, we cloned the rabbit RAG locus and characterized the coding regions of the genomic RAG-1 and RAG-2. In addition, we sequenced cDNAs encompassing the RAG-2 coding region, part of the RAG-2 5' untranslated region and a 967 bp fragment of cDNA from the RAG-1 coding region. Northern analysis revealed a RAG-1 mRNA of 6.6 kb which is similar in size to the RAG-1 mRNA reported previously for other species, and a major species of RAG-2 mRNA of 4.4 kb, which is larger than that from the mouse (2.2 kb). Analysis of the genomic clones showed that, as in other species, the RAG-1 and RAG-2 genes are oriented so as to be convergently transcribed. The DNA sequence analysis showed that the rabbit RAG-1 coding region is 91, 85 and 72% identical to human, mouse and chicken, respectively. The deduced RAG-1 protein sequence for rabbit is 93, 90 and 78% identical to human, mouse and chicken. Comparison of the rabbit RAG-2 coding region revealed 90, 87 and 71% identity to human, mouse and chicken, respectively, at the nucleotide level, and 91, 90 and 72% at the protein level. Although there is considerable conservation of sequence between species, we obtained evidence for allelic forms of the rabbit RAG locus both by Southern analyses and by sequencing. A remarkable degree of polymorphism was found in our rabbit colonies, particularly in the region 3' of the rabbit RAG-2 coding region. A 5' cDNA probe hybridized with one or more additional fragments that are not detected with the coding region probes, suggesting that the 5' cDNA sequence results from splicing of one or more upstream exons.
Mol Immunol 1993 Aug
PMID:Recombination activating genes-1 and -2 of the rabbit: cloning and characterization of germline and expressed genes. 835 Aug 72

To investigate the presence of a negative regulatory factor(s) suppressing T-cell receptor alpha-chain (TCR alpha) gene expression in non-T cells, 10 independent cell hybrid clones were generated between mouse T-cell lymphoma EL4 cells (TCR alpha+/beta+) and mouse fibroblast B82 cells. These cell hybrids showed a typical fibroblastic morphology and retained an approximate sum of chromosome numbers derived from both parental cells. No transcripts of the TCR alpha gene were detected in the cell hybrids, although the presence of the rearranged TCR alpha allele from EL4 cells was confirmed. The possibility of involvement of nuclear proteins responsible for the activity of the TCR alpha gene enhancer in the extinction of TCR alpha gene expression in the cell hybrids was examined. Nuclear proteins which bind to the lymphoid enhancer-binding factor 1 (LEF-1) binding motif present in EL4 cells disappeared in the hybrid clones, whereas no significant change was observed in DNA-binding activity of nuclear proteins to a consensus cyclic AMP response element (CRE) and the Ets-1 binding motif between the parental cells and the cell hybrids. No transcripts of the LEF-1 gene were detected in the cell hybrids, despite the retention of the LEF-1 gene and murine chromosomes 3, on which the LEF-1 allele is located, from both parental cells. These results suggest that a trans-acting negative regulatory factor(s) present in fibroblasts suppresses LEF-1 gene expression and that suppression of LEF-1 may lead to the extinction of TCR alpha gene expression in the cell hybrids.
Mol Cell Biol 1993 Mar
PMID:Extinction of T cell receptor alpha-chain gene expression accompanied by loss of the lymphoid enhancer-binding factor 1 (LEF-1) in murine somatic cell hybrids. 838 79

We used the anchored-polymerase chain reaction (A-PCR) procedure to study human TCR transcripts derived from a variety of polyclonal T cell populations. In this series of experiments, 31 'unusual' cDNAs, which do not include exclusively V-J-C, J-C or 5'C genomic sequences, were identified. Ten of these were found to represent distinct types of alternatively spliced TCR alpha transcripts whose structure is derived from unusual splicing of one, two or even three intervening intronic sequences. The splicing events led to either conservation of a novel exon in the mRNA structure (designated aE1 alpha-aE5 alpha) between the V-J and C segments or to deletion of the 3' V region-J segment. In three cases, the alternatively spliced exons (aE1 alpha-aE3 alpha) interrupt the open translational reading frame of the corresponding V-J alpha segment. Nineteen and two cDNA represent sterile C beta or C delta transcripts, respectively. Their structures are derived from the conservation of a non-translatable exon, aE1 beta or aE1 delta, which is precisely spliced at the 5' end of the corresponding C exon sequences. Interestingly, the 3' region of the aE1 beta sequence is homologous to the murine C beta 0 exon. Together, these results led to the characterization of nine novel exons in the TCR alpha, beta and delta loci.
Mol Immunol 1993 Apr
PMID:Alternatively spliced T cell receptor transcripts expressed in human T lymphocytes. 838 65

The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing.
Mol Cell Biol 1993 Mar
PMID:T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates. 844 6

Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer.
Mol Cell Biol 1996 Mar
PMID:Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties. 862 75

T cell specificity is determined by the combinatorial association of specific variable (V), diversity (D), and junctional (J) regions. Clones of T cells (clonality) can occur, in the blood or in tissue, after proliferation of activated T cells. Determining clonality in mutation assays is necessary to distinguish between mutants and mutational events. We have developed a novel approach to determine clonality among T cell isolates, using restriction digests of PCR-amplified cDNA of the T cell receptor beta gene. The T cell receptor beta gene was PCR-amplified by use of a consensus primer, beginning from a cell pellet of 2,000-5,000 cells or from extracted RNA. This TCR (T cell receptor) beta chain PCR product can also be directly sequenced, allowing simple and easy identification of Vbeta and CDR3 sequence from a small number of cells. The utility of this method is demonstrated by PCR, restriction digest, and sequencing of the TCR beta cDNA from eight T cell clones isolated from 2 individuals. A clone of three identical isolates (one 3-mer) and a clone of two identical isolates (one 2-mer) were determined from restriction digests using two different enzymes. This new method is an easier and more rapid way of determining clonality than traditional methods, e.g., Southern blotting.
Environ Mol Mutagen 1996
PMID:TCR beta PCR from crude preparations for restriction digest or sequencing. 862 46

Many autoimmune diseases are associated with specific class II MHC alleles; however, this association is not complete. One explanation for the variable expression of disease in susceptible individuals is that variability in the TCR repertoire may alter the potential to generate pathogenic autoreactive T cells. The current study was undertaken to examine the possibility that MHC and background heterozygosity, which is the norm in the outbred human population, alters the expressed TCR repertoire and, if so, whether this has an impact on peptide recognition and antigenic specificity. We, therefore, systematically analysed the beef insulin-specific TCR repertoire in inbred BALB/c mice before and after introduction of MHC heterozygosity (BALB/c x BALB.K)F1 mice, or MHC and background gene heterozygosity (BALB/c x A/J)F1 mice. We show that T cells from all three repertoires are predominantly Ad-restricted and recognize the same immunodominant peptide. Despite this, the beef insulin-specific TCR repertoires in F1 mice differ from those seen in BALB/c mice with the most dramatic changes seen in (BALB/c x A/J)F1 mice. These changes are accompanied by subtle differences in the antigenic specificity of the T cells. The results demonstrate that both MHC and background gene heterozygosity affect TCR repertoire selection, suggesting that the variable expression of autoimmune disease in individuals with a susceptible MHC allele may result, in part, from variability in the TCR repertoire introduced by this heterozygosity.
Mol Immunol 1995 Dec
PMID:Both MHC and background gene heterozygosity alter T cell receptor repertoire selection in an antigen-specific response. 864 5

The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCR alpha chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCR alpha chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the alpha chains were derived. Cross-specificity analysis using two antigen-binding TCR alpha chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCR alpha-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding.
Mol Immunol 1995 Dec
PMID:A phage display system for detection of T cell receptor-antigen interactions. 864 8


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