RESUMO
Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a lambda cDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.
Assuntos
AMP Cíclico/fisiologia , Regulação da Expressão Gênica , Glucocorticoides/fisiologia , Linfócitos T/análise , Animais , Northern Blotting , Clonagem Molecular , Colforsina/farmacologia , Cicloeximida/farmacologia , Camundongos , RNA Mensageiro/biossíntese , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Using cDNA cloning techniques we previously identified a set of genes induced by glucocorticoids and cAMP in murine T-lymphocytes. We report here the sequence of one of these cDNA clones (clone 4.2), renamed here as glucocorticoid-induced receptor (GIR), which encodes a potential new member of the family of receptors that couple to G-proteins. Several different forms of cDNA for this gene were isolated and shown to correspond to multiple mRNA species in lymphoid cells using an RNase protection assay. The cDNA clone corresponding to the most abundant form of GIR mRNA encodes a precursor protein of 423 amino acids, with a putative signal peptide of 17 amino acids. A hydropathy plot reveals the presence of seven hydrophobic regions, with significant similarities to other G-protein-coupled receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Regulação da Expressão Gênica , RNA Mensageiro/genética , Receptores de Superfície Celular/genética , Receptores de Neurotransmissores/genética , Linfócitos T/ultraestrutura , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Proteínas de Ligação ao GTP/metabolismo , Camundongos , Dados de Sequência Molecular , Família Multigênica/genética , Especificidade de Órgãos , Conformação Proteica , Receptores de Taquicininas , Homologia de Sequência do Ácido NucleicoRESUMO
Glucocorticoids induce dramatic biochemical and morphological changes in lymphocytes through an unknown process that requires RNA and protein synthesis. In order to identify genes involved in this response, we previously isolated 11 cDNA clones from the murine WEHI-7TG thymoma cell line that correspond to mRNAs induced by glucocorticoids. We now report the isolation of two new cDNA clones whose gene expression is regulated by glucocorticoids in WEHI-7TG cells. We further characterize the two new cDNA clones, as well as those described previously, by examining the response of each of the corresponding mRNAs to glucocorticoids in murine thymocytes. With the exception of two, all cDNAs correspond to genes that are induced by glucocorticoids in murine thymocytes within 4 h of treatment. We previously identified two of the cDNAs as the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein. We have now identified four additional cDNA clones that correspond to the genes for calmodulin, mitochondrial phosphate carrier protein, immunoglobulin (Ig)-related glycoprotein (GP-70), and the 70 kilodalton autoantigen for Lupus and Graves diseases. Two other cDNA clones represent previously undescribed genes: one shares a high similarity to known sequences for the family of G-protein-coupled receptors and the other to a human placental-specific protein, PP11. Another cDNA appears to contain sequences for an unknown gene and the remnants of a mouse transposon. ETn. The remaining clones represent new, unidentified genes induced by glucocorticoids in murine thymocytes and in the WEHI-7TG cell line.