Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The extracellular domain of the PRL receptor (PRL-R) is composed of two subdomains of approximately 100 amino acids, S1 and S2. To explore the functional significance of these subdomains in PRL binding and signal transduction, deletion mutants of S1 or/and S2 subdomains were constructed. We report here the inability of each of these mutant receptor forms to bind PRL after expression in COS-7 cells. We also studied the abilities of these different mutant receptors to respond to hormonal stimulation after transfection of each mutant complementary DNA into CHO-K1 cells along with a chimeric gene containing the promoter of a milk protein gene (beta-lactoglobulin) fused to chloramphenicol acetyltransferase coding sequence. Somewhat unexpectedly, a constitutively (PRL-independent) mutant form of the PRL-R was obtained after deletion of the S2 subdomain. Moreover, we analyzed, in CHO-K1 cells, the biological activity of chimeric receptors constructs in which each subdomain sequence was replaced by an unrelated, but coding, sequence of foreign protein, and we confirmed a specific requirement for the S1 sequence in the constitutive activity. In contrast, the S2 subdomain produced an inhibitory effect on S1 constitutive activity. Cotransfection experiments with the wild-type receptor and the constitutive mutant receptor provided evidence that the wild-type receptor was able to inhibit the constitutive activity of the deleted mutant. Furthermore, in the mouse mammary epithelial cell line HC11, the constitutive PRL-R form was able to induce transcription of the beta-casein gene in the absence of PRL. These results suggest a complex signal transduction process that implicates each extracellular PRL-R subdomain. Possible mechanisms for the constitutive effect are discussed.
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PMID:Development of a constitutively active mutant form of the prolactin receptor, a member of the cytokine receptor family. 883 44

Steroid hormone receptors and Stat factors comprise two distinct families of inducible transcription factors. Activation of a member of each family, namely the glucocorticoid receptor by glucocorticoids and Stat5 by prolactin, is required for the efficient induction of the expression of milk protein genes in the mammary epithelium. We have studied the mode of interaction between Stat5 and the glucocorticoid receptor in the activation of beta-casein gene transcription. The functional role of potential half-palindromic glucocorticoid receptor-binding sites mapped previously in the promoter region was investigated. beta-Casein gene promoter chloramphenicol acetyltransferase constructs containing mutations and deletions in these sites were tested for their responsiveness to the synergistic effect of prolactin and dexamethasone employing COS-7 cells or HC11 mammary epithelial cells. Synergism depended on promoter regions containing intact binding sites for the glucocorticoid receptor and Stat5. The carboxyl-terminal transactivation domains of Stat5a and Stat5b were not required for this synergism. Our results suggest that in lactogenic hormone response elements glucocorticoid receptor molecules bound to nonclassical half-palindromic sites gain competence as transcriptional activators by the interaction with Stat5 molecules binding to vicinal sites.
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PMID:Promoter-dependent synergy between glucocorticoid receptor and Stat5 in the activation of beta-casein gene transcription. 925 24

Milk protein gene expression in mammary epithelial cells is regulated by interactions of the cells with each other and with extracellular-matrix components, and by the lactogenic hormones. Cell-cell and cell-extracellular-matrix interactions confer a state of competence to HC11 mammary epithelial cells. Cellular confluence and matrix deposition are prerequisites for the lactogenic hormone induction of, for example, beta-casein synthesis. We have studied how these cellular interactions influence transcription factor activity. Proximal and distal regulatory elements have been identified in the DNA of the beta-casein gene promoter that confer transcriptional induction to the lactogenic hormones in competent cells. A region located between positions -221 and -170 of the rat beta-casein promoter contains overlapping binding sites for DNA binding factors with positive and negative regulatory activity. A construct containing 221 nt of 5' promoter sequences linked to a chloramphenicol acetyltransferase (CAT) reporter gene and transfected into HC11 cells has low constitutive expression and is strongly inducible. Deletion of the sequences to -183 results in an increase in both constitutive and induced expression. Mutations in or deletion of the region from -183 to -170 abolish promoter activity. A sequence-specific single-stranded DNA binding transcriptional repressor (STR), composed of two proteins, binds to the upper strand of the -194 to -163 fragment and negatively regulates transcription. STR also recognizes the 5' untranslated region of the beta-casein mRNA and is sequestered into the cytoplasm by RNA after lactogenic hormone induction. Sequestration by RNA allows an activator to bind to the fragment -183 to -170. This activator has been identified as SARP, a sequence-specific single-stranded DNA activator region binding protein. The binding site of SARP is found both in the upper and the lower strands of this fragment. SARP has no affinity for RNA. It enhances transcription of a promoter construct containing rat beta-casein promoter sequences from -183 to -1 and of a heterologous promoter containing multimerized copies of the -194 to -163 fragment in a lactogenic-hormone-independent manner. Mutations between positions -183 and -170, which result in a loss of promoter activity, also prevent SARP from binding to the DNA. Confluence of HC11 cells up-regulates the DNA binding activity of SARP. High SARP activity is also detected in mammary gland cells of lactating mice and is regulated by suckling. Withdrawal of pups from their lactating mothers results in a rapid decrease of SARP activity. We have purified SARP from the lactating mammary tissue of sheep and have identified proteins of 28 and 35 kDa.
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PMID:Regulation of gene expression in mammary epithelial cells by cellular confluence and sequence-specific DNA binding factors. 951 16


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