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 LpS1 genes of the sea urchin Lytechinus pictus are activated early in development in aboral ectoderm cells. They therefore have ontogenic properties similar to their counterparts in Stronglyocentrotus purpuratus, the Spec genes. Both gene families encode proteins belonging to the calmodulin superfamily as evidenced by the presence of distinct EF-hand (helix-loop-helix) domains. The presence of eight EF-hand domains in LpS1 proteins suggests that the LpS1 genes arose from a duplication of an ancestral Spec-like gene. The LpS1 genes were further analyzed to increase our understanding of the mechanisms underlying their evolution and activation in aboral ectoderm cells. Genomic DNA blot analysis showed two LpS1 genes, LpS1 alpha and LpS1 beta, which did not appear to be closely linked. LpS1 genomic clones were isolated by screening an L. pictus genomic library with an LpS1 cDNA clone, and partial gene structures for both LpS1 alpha and LpS1 beta were constructed. These revealed internal duplication of the LpS1 genes that accounted for the eight EF-hand domains in the LpS1 proteins. Duplication of exon 1 in both genes suggested four different LpS1 proteins could be derived from the LpS1 genes. Primer extension to map the transcriptional initiation sites of the LpS1 genes and sequencing analysis showed there was little in common among the 5'-flanking regions of the LpS1 and Spec genes except for the presence of a binding site for the transcription factor USF. A sea urchin gene-transfer expression system showed that 762 base pairs (bp) of 5'-flanking DNA and 17 bp of 5'-untranslated leader sequence of the LpS1 beta gene were sufficient for correct temporal and spatial expression of reporter chloramphenicol acetyltransferase and lacZ genes in sea urchin embryos. Deletions at the 5' end to either 511 or 368 bp resulted in a 3-4 fold decrease in chloramphenicol acetyltransferase activity and disrupted the exclusive activation of the lacZ gene in aboral ectodermal cells. Based on a lineage analysis among the LpS1 and Spec gene families and other related genes, we propose a model in which LpS1 genes evolved from a series of duplications of an ancestral Spec-like gene.
 ...
PMID:Structure and promoter activity of the LpS1 genes of Lytechinus pictus. Duplicated exons account for LpS1 proteins with eight calcium binding domains. 203 96

The gene encoding human fast skeletal muscle troponin C (TnC) was cloned, mapped, and sequenced. The locations of intron positions in this gene were compared to those in the related genes for mouse slow skeletal TnC and vertebrate and nonvertebrate calmodulins. We detected strikingly similar purine-rich DNA sequences on the coding strand in the basal promoter of the genes for fast and slow troponin C and chicken calmodulin II which may represent conserved regulatory elements in genes of the vertebrate troponin C/calmodulin gene family. We mapped the transcriptional start site of the gene and analyzed the expression of TnC test genes in the myogenic cell lines C2, L8, and H9c2(2-1) and in the human fibroblast line HuT12. Constructs comprising 4.7 or 6.2 kilobase pairs of 5'-flanking sequence (including the genuine transcriptional start site) upstream of the chloramphenicol acetyltransferase gene as reporter expressed the hybrid gene in C2 cells but not in nonmuscle cells. Surprisingly, no expression was found in cell lines L8 and H9c2(2-1) despite the fact that all three muscle cell lines vigorously express the endogenous TnC fast mRNA after differentiation. The discrepancy between the expression of endogenous genes and the test gene in these cell lines indicates different requirements for regulatory elements in different myogenic cells.
 ...
PMID:Cloning, structural analysis, and expression of the human fast twitch skeletal muscle troponin C gene. 237 3

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.
 ...
PMID:An isoform of transcription factor CREM expressed during spermatogenesis lacks the phosphorylation domain and represses cAMP-induced transcription. 780 53

Engagement of the T cell receptor for antigen activates phospholipase C resulting in an increase in intracellular free calcium concentration ([Ca2+]i) and activation of protein kinase C (PKC). Increased [Ca2+]i activates Ca2+/calmodulin-dependent kinases including the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM-K II), as well as calcineurin, a type 2B protein phosphatase. Recent studies have identified calcineurin as a key enzyme for interleukin (IL)-2 and IL-4 promoter activation. However, the role of CaM-K II remains unknown. We have used mutants of these kinases and phosphatases (gamma B*CaM-K and delta CaM-AI, respectively) to explore their relative role in cytokine gene transcription and their interactions with PKC-dependent signaling systems. gamma B*CaM-K and delta CaM-AI, known to exhibit constitutive Ca(2+)-independent activity, were cotransfected (alone or in combination) in Jurkat T cells with a plasmid containing the intact IL-2 promoter driving the expression of the chloramphenicol acetyltransferase reporter gene. Cotransfection of gamma B*CaM-K with the IL-2 promoter construct downregulated its transcription in response to stimulation with ionomycin and phorbol myristate acetate (PMA). The inhibitory effect of CaM-K II on IL-2 promoter was associated with decreased transcription of its AP-1 and NF-AT transactivating pathways. Under the same conditions, delta CaM-AI superinduced IL-2 promoter activity (approximately twofold increase). When both mutants were used in combination, gamma B*CaM-K inhibited the induction of the IL-2 promoter by delta CaM-AI. Similar results were obtained when a construct containing the IL-4 promoter also was used. gamma B*CaM-K also downregulated the activation of AP-1 in response to transfection with a constitutively active mutant of PKC or stimulation with PMA. These results suggest that CaM-K II may exert negative influences on cytokine gene transcription in human T cells, and provide preliminary evidence for negative cross-talk with the calcineurin- and PKC-dependent signaling systems.
 ...
PMID:Calcium/calmodulin-dependent protein kinase II downregulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells. 786 38

We describe here a strategy for introducing simultaneous, independent gene replacements into the Trypanosoma cruzi chromosome. The goal of this study was to use two linear DNA fragments to simultaneously replace the CalA2 calmodulin and FUS1 ubiquitin-fusion genes with the neomycin resistance (neo(r)) and chloramphenicol acetyltransferase (CAT) genes, respectively. One clone (D6), of thirty G418-resistant clones analyzed, carried the desired dual gene replacement. CDNA sequence analysis indicated that the CAT mRNA was accurately trans-spliced using the previously identified FUS1 mini-exon addition site. However, DNA sequence analysis of the intergenic sequence immediately upstream of the neo(r) gene in clone D6 identified a mutation which altered the pattern of trans-splicing of the neo(r) mRNA. Possible effects of this mutation on 3' splice acceptor site selection are discussed.
 ...
PMID:Analyzing expression of the calmodulin and ubiquitin-fusion genes of Trypanosoma cruzi using simultaneous, independent dual gene replacements. 818 27

Membrane depolarization stimuli (high potassium concentration and veratridine) increased neuropeptide Y (NPY) mRNA abundance time-dependently, without a change in beta-actin mRNA level, in NG108-15 and PC12 cells. Although the induction by veratridine was blocked completely by tetrodotoxin, the induction by potassium was suppressed minimally. Voltage-dependent Ca channel blockers and calmodulin antagonists inhibited the increases by both depolarization stimuli completely, suggesting involvement of Ca2+/calmodulin-dependent kinases (CaM kinases). Transient assay using chloramphenicol acetyltransferase reporter genes containing the rat NPY gene promoter indicated that membrane depolarization and Ca entry stimulate transcription of the NPY gene. The depolarization-induced transactivation was also blocked by CaM kinase inhibitors. The 200-bp 5'-upstream region (-344/-145) was localized as a Ca2+/ calmodulin-responsive element (CaMRE), which confers depolarization-induced transactivation. It is interesting that this CaMRE did not contain the canonical Ca-responsive elements such as CRE, SRE, NF-AT, or the C/EBP beta-binding site and was separated from a 64-bp cyclic AMP/ phorbol 12-myristate 13-acetate-responsive element (-144/-81). These findings suggested that membrane depolarization regulates the NPY gene transcription positively through the unique CaMRE by activation of CaM kinases following Ca entry through L-type Ca channels.
 ...
PMID:Ca2+/calmodulin-dependent transcriptional activation of neuropeptide Y gene induced by membrane depolarization: determination of Ca(2+)- and cyclic AMP/phorbol 12-myristate 13-acetate-responsive elements. 878 4

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor whose ability to bind hormone is thought to be dependent on association with the 90-kDa heat shock protein (hsp90). In the present study, we have generated a novel form of the GR, in which the receptor remains complexed to hsp90 but has lost its ability to bind hormone, by treatment of intact cells with the calmodulin (CaM) antagonist phenoxybenzamine (POBA). Treatment of these cells, mouse L929 cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter construct, with increasing concentrations of POBA resulted in a concentration-dependent inhibition of dexamethasone (Dex)-induced CAT gene expression, with 100 microM POBA resulting in approximately 80% inhibition. This inhibitory effect of POBA was markedly reduced if POBA was added after a short incubation with Dex, suggesting that the primary effect of POBA was on hormone-induced transformation of the GR. Using a subcellular fractionation technique, POBA inhibition of CAT gene expression was found to correlate with an inhibition of Dex-induced GR nuclear translocation. However, inhibition of translocation was not the primary effect of POBA on the GR signal pathway, as POBA was found to reduce GR hormone-binding capacity after treatment of intact cells. The inhibitory effect of POBA on hormone-binding function correlated closely with the inhibitory effect of this drug on CAT gene expression and was not due to an oxidation of sulfhydryl groups, a condition known to reduce GR hormone-binding capacity. Incubation of cytosols from untreated cells with POBA did not decrease GR steroid-binding capacity, demonstrating that this inhibitory effect was not the result of a competitive antagonism at the ligand-binding site. Quantitation of GR protein in the cytosols of POBA-treated cells revealed that the decrease in steroid-binding function was not due to a loss of GR protein. Surprisingly, the amount of GR-bound hsp90 was also unaltered in response to POBA. Taken together, the above observations provide evidence for a novel state of the GR within intact cells in which hsp90 interaction is but one step in the generation or maintenance of hormone-competent receptors. In addition, these results point to the potential use of POBA, and possibly other CaM inhibitors, as antagonists of steroid receptor actions.
 ...
PMID:In vivo evidence for the generation of a glucocorticoid receptor-heat shock protein-90 complex incapable of binding hormone by the calmodulin antagonist phenoxybenzamine. 883 41

Mitochondrial biogenesis can occur rapidly in mammalian skeletal muscle subjected to a variety of physiological conditions. However, the intracellular signal(s) involved in regulating this process remain unknown. Using nuclearly encoded cytochrome c, we show that its expression in muscle cells is increased by changes in cytosolic Ca2+ using the ionophore A23187. Treatment of myotubes with A23187 increased cytochrome c mRNA expression up to 1.7-fold. Transfection experiments using promoter-chloramphenicol acetyltransferase constructs revealed that this increase could be transcriptionally mediated since A23187 increased chloramphenicol acetyltransferase activity by 2.5-fold. This increase was not changed by KN62, an inhibitor of Ca2+/calmodulin-dependent kinases II and IV, and it was not modified by overexpression of protein kinase A and cAMP response element-binding protein, demonstrating that the A23187 effect was not mediated through Ca2+/calmodulin-dependent kinase- or protein kinase A-dependent pathways. However, treatment of myotubes with staurosporine or 12-O-tetradecanoylphorbol-13-acetate reduced the effect of A23187 on cytochrome c transactivation by 40-50%. Coexpression of the Ca2+-sensitive protein kinase C isoforms alpha and betaII, but not the Ca2+-insensitive delta isoform, exaggerated the A23187-mediated response. The short-term effect of A23187 was mediated in part by mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) since its activation peaked 2 h after A23187 treatment, and cytochrome c transactivation was reduced by PD98089, a mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor. These results demonstrate the existence of a Ca2+-sensitive, protein kinase C-dependent pathway involved in cytochrome c expression and implicate Ca2+ as a signal in the up-regulation of nuclear genes encoding mitochondrial proteins.
 ...
PMID:Calcium-dependent regulation of cytochrome c gene expression in skeletal muscle cells. Identification of a protein kinase c-dependent pathway. 1009 7

The neurohypophyseal nonapeptide Arg8 vasopressin (AVP) promotes differentiation of cultured L6 and L5 myogenic cell lines and mouse primary satellite cells. Here, we investigated the molecular mechanism involved in the induction of the myogenic program by AVP. In L6 cells, AVP treatment rapidly induces Myf-5, myogenin, and myocyte enhancer factor 2 (MEF2) mRNAs, without affecting the expression of known myogenic growth factors such as IGF-I, IGF-II, or their receptors. In the presence of cycloheximide, AVP up-regulates the expression of MEF2, but not of myogenin, indicating that the synthesis of a protein intermediate is not necessary for MEF2 induction. Notably, AVP treatment activates a calcium/calmodulin kinase signaling pathway that induces cytosolic compartmentalization of the histone deacetylase 4, a mechanism related to the transcriptional activation of MEF2. The activity of chloramphenicol acetyltransferase reporter constructs carrying the Myo184 and Myo84 fragments of the myogenin promoter is also induced by AVP. Mutation of the MEF2 site completely abolishes the response to AVP, whereas deletion of the E1 site present in pMyo84 does not impair this response. Together, these results show that AVP induces myogenic differentiation through the transcriptional activation of MEF2, a mechanism that is critical for myogenesis.
 ...
PMID:AVP induces myogenesis through the transcriptional activation of the myocyte enhancer factor 2. 1204 25

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.
 ...
PMID:Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion. 1555 68


1 2 Next >>