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)

The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident. However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes.
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PMID:Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative. 173 26

We present evidence that CRE-BP1 binding to the cyclic AMP (cAMP) response element (CRE) is a transcriptional activator. Transcriptional activation was assayed by cotransfection into CV-1 cells of a CRE-BP1 expression plasmid together with a reporter plasmid in which the thymidine kinase promoter and four tandem repeats of CRE were linked to the chloramphenicol acetyltransferase (CAT) gene. Cotransfection with the CRE-BP1 expression plasmid caused an 8-fold stimulation of CAT activity, while cotransfection with the plasmids to express CRE-BP1 and c-Jun induced a 32-fold stimulation of CAT activity, suggesting that a heterodimer of CRE-BP1 with c-Jun is a stronger trans-activator than a homodimer of CRE-BP1. By using a series of deletion and point mutants of CRE-BP1 in this cotransfection assay, two functional domains of CRE-BP1 were identified: the putative metal finger structure in the amino-terminal region and the leucine zipper motif linked to a cluster of basic amino acids in the carboxyl-terminal region. The former was a transcriptional activation domain in the absence of c-Jun. The latter was a DNA-binding domain, and was essential in both the presence and absence of c-Jun.
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PMID:Identification of the functional domains of the transcriptional regulator CRE-BP1. 183 93

To isolate transcription factors important in the regulation of the human interleukin-3 (IL-3) gene, we screened a lambda gt11 cDNA library, constructed from phytohemagglutinin-stimulated human T-cell RNA, with a probe containing regulatory sequences in the upstream region of the IL-3 gene (located from bp -165 to -128 and referred to as the DNase I footprint A region). We isolated a 0.96-kb cDNA that encoded a basic amino acid domain and a leucine zipper domain and used the "rapid amplification and cloning of 3' ends" technique to isolate the 3' half of the cDNA clone, generating a 1.9-kb full-length cDNA clone. Using in vitro-translated protein, which we call NF-IL3A, we defined the IL-3 promoter sequences bound by NF-IL3A in DNase I footprinting assays as TAATTACGTCTG and, using gel shift assays, defined ATTACG as the minimal sequence required for binding of NF-IL3A in vitro. Proteins that bind to the NF-IL3A binding site are found in both unstimulated and stimulated T-cell lines in similar amounts, although the level of NF-IL3A mRNA increases after T-cell activation in several mature T-cell lines. The NF-IL3A protein is nearly identical to a recently identified transcriptional repressor protein, E4BP4, and NF-IL3A binds specifically to regulatory sequences in both the adenovirus E4 promoter and the human gamma interferon promoter. Cotransfection experiments demonstrate that introduction of an expression vector containing the NF-IL3A cDNA into resting T cells transactivates IL-3 promoter-chloramphenicol acetyltransferase gene plasmids that contain the A region; this effect requires the presence of an intact NF-IL3A binding site. One or more copies of the A region also confer NF-IL3A responsiveness on a heterologous promoter in T cells. NF-IL3A appears to play an important role in the expression of IL-3 by T cells.
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PMID:Molecular cloning and characterization of NF-IL3A, a transcriptional activator of the human interleukin-3 promoter. 756 58

Heat shock factors (HSF) are the transcriptional activators of the heat shock response. The conversion of constitutively expressed HSF to a form that can bind DNA requires the trimerization of the protein, involving leucine zipper interactions as shown for yeast, Drosophila, chicken and human HSFs. Like other metazoan HSFs, the endogenous Arabidopsis HSF displays heat shock-inducible DNA-binding activity in gel retardation assays. The heat shock-inducible binding of a recombinant Arabidopsis HSF (ATHSF1) expressed in Arabidopsis plants suggests that ATHSF1 is the major heat shock factor regulating the heat stress response. However, on transient expression in Drosophila and human cells, ATHSF1 fails to exhibit proper regulation, as demonstrated by constitutive binding to DNA, and by constitutive expression of a chloramphenicol acetyltransferase (CAT) reporter gene under the control of the Drosophila hsp70 promoter. These results suggest that the regulation of ATHSF1 is normally dependent on a specific factor that inhibits the DNA-binding and transcriptional activities under non-heat shock conditions.
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PMID:Arabidopsis heat shock factor is constitutively active in Drosophila and human cells. 765 36

Stromelysins, which are the metalloproteinases with the widest substrate specificities, play a critical role in tumor invasion and metastasis. We have previously reported an element (SPRE) of the stromelysin promoter located between nucleotides -1221 and -1203 that is necessary and sufficient for the control of stromelysin gene expression by mitogenic activation, which induces a nuclear activity that binds to this sequence. Using a concatenated probe with several copies of this element to screen a lambda gt11 cDNA expression library from mouse Swiss 3T3 fibroblasts, we report here the molecular cloning of a cDNA coding for a novel protein (SPBP) of 937 amino acids that binds to this element and has several features of a transcription factor, such as a putative leucine zipper region, a nuclear localization signal, and a basic domain with homology to the DNA-binding domains of Fos and Jun. Evidence that SPBP is at least a critical component of the mitogen-induced SPRE nuclear binding activity is presented here. Furthermore, the transfection of an expression plasmid for SPBP transactivates reporter chloramphenicol acetyltransferase plasmids containing either the full-length stromelysin promoter or a single copy of the SPRE cloned upstream of the herpes simplex virus thymidine kinase minimal promoter. Therefore, the results presented here identify a novel transcription factor critically involved in the control of stromelysin expression.
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PMID:Molecular characterization of a novel transcription factor that controls stromelysin expression. 776 Aug 12

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

Transcriptional activation of the mouse c-fos gene by the adenovirus 243-amino-acid E1A protein requires a binding site for transcription factor YY1 located at -54 of the c-fos promoter. YY1 normally represses transcription of c-fos, and this repression depends on the presence of a cyclic AMP (cAMP) response element located immediately upstream of the -54 YY1 DNA-binding site. This finding suggested that the mechanism of transcriptional repression by YY1 might involve a direct interaction with members of the ATF/CREB family of transcription factors. In vitro and in vivo binding assays were used to demonstrate that YY1 can interact with ATF/CREB proteins, including CREB, ATF-2, ATFa1, ATFa2, and ATFa3. Structure-function analyses of YY1 and ATFa2 revealed that the C-terminal zinc finger domain of YY1 is necessary and sufficient for binding to ATFa2 and that the basic-leucine zipper region of ATFa2 is necessary and sufficient for binding to YY1. Overexpression of YY1 in HeLa cells resulted in repression of a mutant c-fos chloramphenicol acetyltransferase reporter that lacked binding sites for YY1, suggesting that repression can be triggered through protein-protein interactions with ATF/CREB family members. Consistent with this finding, repression was relieved upon removal of the upstream cAMP response element. These data support a model in which YY1 binds simultaneously to its own DNA-binding site in the c-fos promoter and also to adjacent DNA-bound ATF/CREB proteins in order to effect repression. They further suggest that the ATF/CREB-YY1 complex serves as a target for the adenovirus 243-amino-acid E1A protein.
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PMID:Transcriptional repression of the c-fos gene by YY1 is mediated by a direct interaction with ATF/CREB. 776 93

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

When antagonist-occupied steroid receptors have agonist-like effects, the clinical consequences are grave. We present evidence that human progesterone B-receptors (hPRB) when occupied by progesterone antagonists, inappropriately activate transcription by an unusual mechanism that does not require the canonical progesterone response element (PRE). In HeLa cells cotransfected with a PRE-tk-chloramphenicol acetyltransferase reporter and a hPRB expression vector, strong transcription is seen not only when receptors are activated by the agonist R5020, but also in the presence of the three antiprogestins, RU486, ZK112993, and ZK98299. Human PRB occupied by ZK98299 do not bind to a PRE, suggesting that the transcriptional stimulation is independent of DNA binding. Indeed, a tk-chloramphenicol acetyltransferase promoter-reporter lacking the PRE loses transcriptional activation by the agonist, but retains transactivation by the three antagonists. The PRE-independent antagonist-induced transcription requires that hPRB have an intact DNA-binding domain, but hPR target gene specificity is not required, because a hPRB mutant that binds an estrogen response element still activates transcription. It appears that antagonist-occupied hPR activate transcription without binding to a PRE, perhaps by interacting with tethering proteins instead. Even a gene that is not a normal progesterone target could be aberrantly activated. Human cells contain equimolar amounts of hPRB and the N-terminally truncated natural isotype, hPRA. Unlike hPRB, hPRA are not transcriptionally activated by progesterone antagonists. We, therefore, tested the effects of antagonists when the two receptor isotypes are coexpressed and found that A-receptors can annul the inappropriate transcription by B-receptors. Thus, when both receptor forms are present, the hPRA phenotype is dominant. Moreover, pure hPRB/hPRA heterodimers, produced by fos/jun leucine zipper domain-hPR chimeras, also have the inactive transcriptional phenotype of hPRA. Our studies suggest not only that the two hPR isotypes are functionally quite different, but also that some of the agonist-like transcriptional effects of antagonist-occupied B-receptors proceed through novel mechanisms.
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PMID:Antagonist-occupied human progesterone B-receptors activate transcription without binding to progesterone response elements and are dominantly inhibited by A-receptors. 826 57

DNA-binding proteins containing the basic helix-loop-helix (bHLH) domain have been implicated in lineage determination and the regulation of specific gene expression in a number of cell types. By oligonucleotide screening of an adipocyte cDNA expression library, we have identified a novel member of the bHLH-leucine zipper transcription factor family designated ADD1. ADD1 mRNA is expressed predominantly in brown adipose tissue in vivo and is regulated during both determination and differentiation of cultured adipocyte cell lines. ADD1 can function as a sequence-specific transcriptional activator in that it stimulates expression of a chloramphenicol acetyltransferase vector containing multiple ADD1 binding sequences but is unable to activate the myosin light-chain enhancer, which contains multiple binding sites for another bHLH factor, MyoD. ADD1 can also activate transcription through a binding site present in the 5'-flanking region of the fatty acid synthetase gene which is expressed in a differentiation-dependent manner in adipose cells. These data suggest that ADD1 plays a role in the regulation of determination- and differentiation-specific gene expression in adipocytes.
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PMID:ADD1: a novel helix-loop-helix transcription factor associated with adipocyte determination and differentiation. 833 13


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