RESUMEN
We have isolated and characterized the gene coding for the mouse Fc receptor that is termed Fc gamma RIIa. The gene contains five exons and spans approximately 9 kilobases. Unlike most members of the immunoglobulin gene superfamily, this gene utilizes multiple exons to encode its leader peptide. The first exon encodes the hydrophobic region of the signal sequence; the second exon, which contains only 21 base pairs, encodes a segment of the signal peptidase recognition site; and the beginning of the third exon encodes the predicted site of peptidase cleavage. The third and fourth exons each code for immunoglobulin-like extracellular domains. The fifth exon encodes the hydrophobic transmembrane domain and the cytoplasmic tail. Partial characterization of the Fc gamma RIIb gene indicates that it also contains multiple leader exons, including a 21-base-pair exon and two exons coding for homologous immunoglobulin-like extracellular domains. However, the Fc gamma RIIb gene uses four exons to encode its intracytoplasmic region. Analysis using contour-clamped homogeneous electric field (CHEF) gels indicates that the Fc gamma RIIa and Fc gamma RIIb genes are linked within 160 kilobases on mouse chromosome 1.
Asunto(s)
Antígenos de Diferenciación/genética , Familia de Multigenes , Receptores Fc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Exones , Ratones , Datos de Secuencia Molecular , Empalme del ARN , Receptores de IgG , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Terminal deoxynucleotidyl transferase (TdT) is a nuclear enzyme found in early lymphocytes which is thought to increase junctional diversity of TCR and Ig genes by the addition of N regions. TdT is normally found only in immature lymphoid cells and is turned off in both mature B and T cells. To investigate the regulation of TdT gene expression, pre-B and pre-T cells were treated with PMA or three of its analogs and its effects on steady-state TdT mRNA levels determined. Rapid and reversible decline in steady-state TdT mRNA levels was observed within 6 h with PMA. This rapid decline can be blocked by pretreatment of the cells with a relatively selective protein kinase C inhibitor implicating the role of protein kinase C activation in the decline of TdT mRNA. Nuclear run-off studies demonstrate that TdT transcription is rapidly down-regulated within 45 min after PMA treatment, indicating that this regulation occurs mainly at the level of transcription. Furthermore, the TdT mRNA decline is blocked in the presence of cycloheximide, showing that new protein synthesis is required for inactivation of the gene.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Proteína Quinasa C/fisiología , Animales , Linfocitos B/enzimología , Linfocitos B/metabolismo , Unión Competitiva , Línea Celular , Cicloheximida/farmacología , ADN Nucleotidilexotransferasa/genética , Activación Enzimática/efectos de los fármacos , Humanos , Activación de Linfocitos/efectos de los fármacos , Ratones , Proteína Quinasa C/antagonistas & inhibidores , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
To characterize the T cell receptor repertoire in an immune response in which the Ia and nominal antigenic determinants are defined and limited, we have cloned and sequenced the expressed receptors from four independent, beef insulin-specific T cell lines from C57BL mice. Each of these lines responded to beef but not to the pork insulin, thus defining the nominal antigenic determinant recognized. Furthermore, each of these lines could only be presented antigen by B6 but not mutant B6.C-H-2bm12 antigen-presenting cells, thus defining the requisite Ia recognition or antigen-association site. In spite of this functional similarity in ligand specificity, each of these T cell lines was found to use different V alpha and V beta gene segments. Moreover, structural comparisons of implied protein sequences of each of these receptors showed no stretches of conserved amino acid residues that could be implicated in ligand interaction. However, the V alpha genes used by these four clones appeared considerably more homologous to each other than were their V beta genes.
Asunto(s)
Insulina/inmunología , Ratones Endogámicos C57BL/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Línea Celular , Ratones , Ratones Endogámicos C57BL/genética , Fragmentos de Péptidos/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta , Homología de Secuencia de Ácido NucleicoRESUMEN
In order to characterize the variability of the expressed human T-cell receptor (TCR) beta-chain repertoire and contrast this variability to the known murine beta-chain repertoire, 15 independent complementary DNA (cDNA) clones containing TCR beta-chain variable region (V beta) genes were isolated from a human tonsil cDNA library. The nucleotide and derived amino acid sequences of these 15 V beta genes were analyzed together with 7 previously defined sequences. Fifteen different human V beta genes could be identified from 22 independent sequences. By means of DNA hybridization and sequence homology comparisons, it was possible to group these 15 genes into ten distinct V beta subfamilies, each containing from one to seven members. Minimal polymorphism was noted between individuals, except in multimember subfamilies. The amino acid sequences of these genes contain conserved amino acids that are also shared by murine TCR V beta genes and immunoglobulins; no features were found that distinguish human V beta genes from their murine counterparts. Evaluation of secondary structure showed that maximum variability coincides with generally hydrophilic portions of the amino acid sequence, while specific hydrophobic regions were conserved in all V beta genes examined.