Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hereditary 1,25-dihydroxyvitamin D3-resistant rickets is a human syndrome that arises as a result of heterogeneous molecular defects in the vitamin D3 receptor. Recent studies have identified single unique point mutations within the second or third exons that encode the DNA-binding domain of the vitamin D receptor (VDR) gene in two families with this syndrome. In the experiments reported here, these mutations were introduced into the normal VDR cDNA by site-directed mutagenesis and the mutant products evaluated for hormone, nuclear, and DNA-binding characteristics. Each mutant VDR was expressed in COS-1 cells at equivalent levels, and saturation analysis of cell cytosol revealed normal affinity for the 1,25-dihydroxyvitamin D3 hormone. Incubation of transfected cells with radiolabeled hormone followed by lysis and extraction suggests a lowered salt dependence for solubilization of the mutant VDR. Concomitantly, mutant receptors exhibited reduced affinity for immobilized calf thymus DNA. While cotransfection of the wild type receptor together with a vitamin D-inducible (osteocalcin) chloramphenicol acetyltransferase reporter gene construction in CV-1 cells resulted in strong induction by 1,25-dihydroxyvitamin D3, neither mutant receptor was capable of directing significant activity either as a function of receptor or hormone concentration. These data suggest that the unique point mutations identified in each of these two families are responsible not only for the phenotype originally ascribed to the abnormal receptor but also severely compromise each protein's ability to activate transcription.
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PMID:Mutant vitamin D receptors which confer hereditary resistance to 1,25-dihydroxyvitamin D3 in humans are transcriptionally inactive in vitro. 255 49

The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.
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PMID:Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae. 297 53

Insulin increases expression of somatostatin-chloramphenicol acetyltransferase (CAT) constructs 10-fold and thymidine kinase-CAT constructs 5-fold in GH4 cells. These responses are similar to our previously reported data on insulin-increased prolactin-CAT expression. They are also observed in HeLa cells and are thus not cell type specific. The evidence suggests that the insulin responsiveness of these genes is mediated by an Ets-related transcription factor. First, linker-scanning mutations and/or deletions of the prolactin, somatostatin, and thymidine kinase promoters suggest that their insulin responsiveness is mediated by the sequence CGGA. This sequence is identical with the response element of the Ets-related transcription factors. Second, CGGA-containing sequences placed at -88 in the delta MTV-CAT reporter plasmid conferred insulin responsiveness to the mammary tumor virus promoter. Third, expression of the DNA-binding domain of c-Ets-2, which acts by blocking effects mediated by Ets-related transcription factors, inhibits the response of these promoters to insulin. Finally, the Ets-related proteins Sap and Elk-1 bind to the prolactin, somatostatin, and thymidine kinase insulin-response elements. An Ets-like element was found in all insulin-sensitive promoters examined and may serve a similar function in those promoters.
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PMID:A consensus insulin response element is activated by an Ets-related transcription factor. 749 46

We previously isolated a cDNA clone encoding interferon consensus sequence-binding protein (ICSBP), a member of the interferon regulatory factor (IRF) family, that binds to the interferon (IFN)-stimulated response element (ISRE) of many IFN-regulated genes. In this investigation, we studied the functional role of ICSBP by transient cotransfection of ICSBP cDNA with IFN-responsive reporter genes into the human embryonal carcinoma cell line N-Tera2. These cells were shown not to express ICSBP or IRF-2, thus allowing functional analysis of transfected cDNAs. Cotransfection of ICSBP into cells treated with retinoic acid or any of the IFNs (alpha, beta, or gamma) repressed expression of a chloramphenicol acetyltransferase reporter driven by the major histocompatibility complex class I gene promoter. Similarly, ICSBP repressed expression of chloramphenicol acetyltransferase reporters driven by the ISREs of the 2'-5' oligoadenylate synthetase, guanylate-binding protein, and ISG-15 genes in IFN-treated cells. The repression was dependent on the presence of the ISRE in the reporter. Deletion analysis showed that the putative N-terminal DNA binding domain of ICSBP by itself is capable of mediating the repression. Using the same cotransfection conditions as for ICSBP, a similar repression of these reporters was observed with IRF-2. Finally, ICSBP repressed the IRF-1-mediated induction of major histocompatibility complex class I and IFN-beta reporters in the absence of IFN or retinoic acid. Taken together, these results suggest that ICSBP is a negative regulatory factor capable of repressing transcription of target genes induced by IFN, retinoic acid, or IRF-1.
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PMID:Interferon consensus sequence-binding protein, a member of the interferon regulatory factor family, suppresses interferon-induced gene transcription. 767 54

A two-hybrid system was used to study interaction in vivo between the nucleocapsid protein (NP) and the phosphoprotein (P) of human parainfluenza virus type 3 (HPIV-3). Two plasmids, one containing the amino terminus of P fused to the DNA-binding domain of the yeast transactivator, GAL4, and the other containing the amino terminus of NP fused to the herpesvirus transactivator, VP16, were transfected in COS-1 cells along with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 DNA-binding sites. A specific and high-affinity interaction between NP and P was observed as measured by the activation of the CAT gene. Mapping of the domains in P (603 amino acids) involved in the association with NP revealed that NH2-terminal 40 and COOH-terminal 20 amino acids are important for such association. Interestingly, a stretch of NH2-terminal amino acids as short as 63-403 interacted with NP more than the wild type, reaching greater than 2.5-fold as measured by the CAT assay. These results suggest that a domain is present in P that negatively regulates its interaction with NP. Deletion of NH2-terminal 40 and COOH-terminal 160 amino acids of NP reduced the CAT activity by more than 95%. These results underscore the important differences between negative strand RNA viruses with respect to interactions between these two viral proteins involved in gene expression.
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PMID:Interaction between the nucleocapsid protein and the phosphoprotein of human parainfluenza virus 3. Mapping of the interacting domains using a two-hybrid system. 775 93

Marek's disease virus (MDV) is an avian herpesvirus that induces a variety of diseases, including T-cell lymphomas, in chickens. In latently infected, transformed lymphoid cells, very few viral transcripts or proteins are detected. We previously described a gene, meq (MDV EcoQ), which is persistently expressed in MDV-transformed tumor samples and cell lines. meq codes for a 339-amino-acid protein with a basic-leucine zipper domain near its N terminus and a proline-rich domain near its C terminus. The basic-leucine zipper domain shows homology with Jun/Fos family proteins, whereas the proline-rich domain resembles that of the WT-1 tumor suppressor protein. These structural features raise the possibility that Meq functions as a transcription factor in regulating viral latency or oncogenesis. In this report, we show that the proline-rich domain is a potent transcription activator when fused to the yeast (Saccharomyces cerevisiae) Gal4(1-147) DNA-binding domain. The transactivation activity maps to the C-terminal 130 amino acids, with the last 33 amino acids essential. In the absence of these 33 amino acids, a two-and-one-half proline-rich repeat structure was found to exhibit repression activity. We further show that Meq is able to dimerize not only with itself but also with c-Jun. Meq/c-Jun heterodimers bind to an AP1-like sequence in the meq promoter region with an affinity much greater than that of Meq/Meq or c-Jun/c-Jun homodimers. Cotransfection chloramphenicol acetyltransferase assays suggest that the Meq/c-Jun heterodimers can up-regulate Meq expression in both chicken embryo fibroblasts and F9 cells. Our data provide the first biochemical evidence that Meq is a transcriptional factor and identify c-Jun as one of Meq's interacting partners.
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PMID:Transactivation activity of Meq, a Marek's disease herpesvirus bZIP protein persistently expressed in latently infected transformed T cells. 776 61

cAMP response element-binding protein (CREB) and modulator protein (CREM) regulate the transcription of cAMP-responsive genes via phosphorylation by cAMP-dependent protein kinase A. Reverse transcription and polymerase chain amplification of RNA from male germ cells identify an alternatively spliced CREM isoform, CREM delta C-G, lacking four exons including those encoding the protein kinase A-regulated phosphorylation domain and the flanking glutamine-rich transcriptional activation domains. CREM delta C-G retains exons that encode the basic-leucine zipper (bZIP) DNA-binding domain, binds to cAMP response elements (CREs), and competitively inhibits binding of CREB and CREM to CREs. Expression of CREM delta C-G inhibits transcription of a CRE-containing chloramphenicol acetyltransferase reporter plasmid induced by endogenous CREB. Antiserum to CREM detects CREM delta C-G in elongated spermatids from rat testis. These observations indicate that CREM delta C-G is a unique form of a competitive negative regulator of CREB-mediated gene transcription expressed in a maturation-dependent manner in haploid germ cells. The developmental specificity of CREM delta C-G suggests that it may play a role in transcriptional regulation during spermatogenesis.
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PMID:An isoform of transcription factor CREM expressed during spermatogenesis lacks the phosphorylation domain and represses cAMP-induced transcription. 780 53

Human progesterone target tissues contain two progesterone receptors: B-receptors (hPRB), which are 933 amino acids in length, and A-receptors (hPRA), which lack the N-terminal 164 amino acids. The two isoforms differ functionally when they are occupied by agonists or antagonists. We postulated that the unique 164-amino acid, B-upstream segment (BUS) is in part responsible for the functional differences between the two isoforms and have constructed a series of hPR expression vectors encoding BUS fused to isolated down-stream functional domains of the receptors. These include the two transactivation domains: activation function-1 (AF1), located in a 90-amino acid segment just up-stream of the DNA-binding domain (DBD) and nuclear localization signal (NLS), and AF2, located in the hormone-binding domain. BUS is a highly phosphorylated domain, and contains the serine residues responsible for the hPRB triplet protein structure. The construct containing BUS-DBD-NLS binds tightly to DNA when aided by accessory nuclear factors. In HeLa cells, BUS-DBD-NLS strongly and autonomously activates transcription of chloramphenicol acetyltransferase (CAT) from a promoter containing two progesterone response elements (PRE2-TATAtk-CAT). Transcription levels with BUS-DBD-NLS are equivalent to those seen with full-length hPRB, and are higher than those seen with hPRA. BUS specifically requires an intact hPR DBD to be transcriptionally active. DBD mutants that cannot bind DNA or whose DNA binding specificity has been switched to an estrogen response element cannot cooperate in BUS transcriptional activity. The function of BUS-DBD-NLS is promoter and cell specific. It does not transactivate a CAT reporter driven by the mouse mammary tumor virus promoter in HeLa cells and poorly transactivates PRE2-TATAtk-CAT in PR-negative T47D breast cancer cells. However, in the breast cancer cells, BUS-DBD-NLS transactivation of PRE2-TATAtk-CAT can be reconstituted by either elevating cellular levels of cAMP or linking BUS and DBD to AF1 or AF2 of hPR, each of which alone is also inactive in these cells. We conclude that hPRB contains a unique third activation function (AF3) located within BUS and requiring the functional DBD of hPR. Depending on the promoter or cell tested, AF3 can activate transcription autonomously, or it can functionally synergize with AF1 or AF2. Autonomous AF3 function may explain the unexpected transactivating actions of antiprogestin-occupied hPRB, an issue of importance in hormone-resistant breast cancers and in tissue-specific agonist-like effects of hormone antagonists.
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PMID:A third transactivation function (AF3) of human progesterone receptors located in the unique N-terminal segment of the B-isoform. 785 52

The steroid/hormone nuclear receptor superfamily comprises several subfamilies of receptors that interact with overlapping DNA sequences and/or related ligands. The thyroid/retinoid hormone receptor subfamily has recently attracted much interest because of the complex network of its receptor interactions. The retinoid X receptors (RXRs), for instance, play a very central role in this subfamily, forming heterodimers with several receptors. Here we describe a novel member of this subfamily that interacts with RXR. Using a v-erbA probe, we obtained a cDNA which encodes a novel 445-amino-acid protein, RLD-1, that contains the characteristic domains of nuclear receptors. Northern (RNA) blot analysis showed that in mature rats, the receptor is highly expressed in spleen, pituitary, lung, liver, and fat. In addition, weaker expression is observed in several other tissues. Amino acid sequence alignment and DNA-binding data revealed that the DNA-binding domain of the new receptor is related to that of the thyroid/retinoid subgroup of nuclear receptors. RLD-1 preferentially binds as a heterodimer with RXR to a direct repeat of the half-site sequence 5'-G/AGGTCA-3', separated by four nucleotides (DR-4). Surprisingly, this binding is dependent to a high degree on the nature of the spacing nucleotides. None of the known nuclear receptor ligands activated RLD-1. In contrast, a DR-4-dependent constitutive transcriptional activation of a chloramphenicol acetyltransferase reporter gene by the RLD-1/RXR alpha heterodimer was observed. Our data suggest a highly specific role for this novel receptor within the network of gene regulation by the thyroid/retinoid receptor subfamily.
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PMID:A novel orphan receptor specific for a subset of thyroid hormone-responsive elements and its interaction with the retinoid/thyroid hormone receptor subfamily. 793 18

The estrogen receptor (ER) typically activates gene transcription by binding to estrogen-responsive elements (EREs). The brain creatine kinase (BCK) promoter is responsive to estrogen but contains no ERE-related sequence. To investigate the mechanism of estrogen induction, we have introduced the estrogen receptor into HeLa cells and primary rat cardiomyocytes and fibroblasts along with 195 bp of BCK promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene. A 10-fold stimulation of CAT activity was observed in the presence of beta-estradiol in both HeLa and rat primary fibroblasts, but no induction was observed in primary rat cardiomyocytes. In contrast, a control vitellogenin gene construct which contains a typical ERE was induced in an ER-dependent manner in all cell types studied. Estrogen induction in HeLa was not sensitive to cycloheximide and was blocked by the ER antagonists tamoxifen and ICI 164,384. Analysis of 5' deletion and linker-scanning mutations indicates sequences between bp -45 and -75 including a TA-rich sequence and a CCAAT sequence to be crucial for stimulation of the BCK promoter by the ER. BCK estrogen induction is dependent on the DNA-binding domain and transactivation domain TAF2 of the ER. However, direct DNA binding is probably not required. Taken together, these results suggest a novel mechanism for ER-mediated gene activation. This mechanism is consensus ERE independent and cell type specific and requires interactions between the ER and molecules capable of interacting with the BCK promoter TA-rich region.
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PMID:A novel, cell-type-specific mechanism for estrogen receptor-mediated gene activation in the absence of an estrogen-responsive element. 793 28


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