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)

Using electroporation-mediated gene transfer, the gene encoding the Slow (S) migrating polypeptide of the maize (Zea mays L.) alcohol dehydrogenase-1 (Adh1) enzyme has been introduced stably and transiently into maize cells containing an endogenous Fast (F) ADH1 electromorph. In stable transformants an 11.5-kb fragment was sufficient to program normal S expression relative to the endogenous F allele. In transient assays, Adh1-S gene constructs lacking the 9 Adh1-S intervening sequences (introns) were expressed at levels 50- to 100-fold less than the intact gene; the presence of intron 1 alone restored levels of gene expression to those found with the intact gene. The last two introns also stimulate Adh1-S expression, but the level is threefold below that of the intact gene. The expression of a chimeric chloramphenicol acetyltransferase (CAT) gene utilizing the 5' promoter and 3' polyadenylation regions of the Adh1 gene was increased 100-fold by the addition of sequences containing the Adh1 intron 1. The Adh1 intron 1 sequences did not stimulate CAT expression when located outside the transcribed region. When located within the transcribed region, the Adh1 intron 1 region efficiently stimulated CAT expression only when located between the promoter and the CAT coding region. A construct containing the Adh1 intron 1 fragment produced 40-fold more mRNA than a construct containing an equivalent cDNA fragment. Both the Adh1 intron 1 and the intron from a second maize gene, Bronze1, stimulated expression from other promoters (cauliflower mosaic virus 35S and nopaline synthase) and of other coding regions (luciferase and neomycin phosphotransferase II) as well. These results indicated that introns increase both Adh1 and chimeric gene expression in maize and the optimal location for such an intron is near the 5' end of the mRNA.
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PMID:Introns increase gene expression in cultured maize cells. 282 68

A recombinant vaccinia virus that expresses the human immunodeficiency virus (HIV) trans-activator (tat) gene was constructed. The tat polypeptide migrated anomalously with an apparent molecular mass of 14 kDa on a sodium dodecyl sulfate-polyacrylamide gel and reacted with polyclonal anti-tat serum. The tat protein was localized predominantly in the cell nucleus despite the absence of other HIV proteins or intranuclear HIV DNA. Additional recombinant vaccinia viruses that contain the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under control of an early vaccinia promoter were constructed. Insertion of the HIV trans-activator-responsive (tar) sequence at the precise start of the CAT mRNA decreased CAT expression slightly. Trans-activation of vaccinia virus-encoded tarCAT failed to occur when CV-1 or HeLa cells were coinfected with the recombinant vaccinia virus expressing tat or when a HeLa cell line containing stably integrated copies of tat was used for infection, indicating the absence of transcriptional or translational effects under these conditions.
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PMID:Use of vaccinia virus vectors to study the synthesis, intracellular localization, and action of the human immunodeficiency virus trans-activator protein. 283 62

A modular system for assaying the activity of transcriptional regulatory signals based on herpes simplex virus (HSV) promoter and terminator sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene has been used to study activation of HSV immediate early (IE) gene expression. Insertion of the SV40 72 base pair (bp) repeat increased mRNA levels by 15-fold thus demonstrating the ability of the HSV IE promoter to respond to a heterologous enhancer. A fragment containing part of the intergenic region located between HSV-2 immediate early (IE) genes-3 and -4/-5 increased mRNA levels by 5-fold in response to transactivation by an HSV virion structural polypeptide. The HSV activator fragment increased mRNA levels by 2-fold in the absence of transactivation indicating that cellular proteins are involved in IE gene expression. From HSV-1/HSV-2 DNA sequence comparisons we previously proposed that a DNA sequence, consensus TAATGARAT, present upstream of all HSV-1 and HSV-2 IE genes was required for the co-ordinate induction of IE genes. We show here that a synthetic oligonucleotide containing TAATGARAT conferred the ability to stimulate CAT activity only on transactivation: two copies of TAATGARAT stimulated expression by 2-fold while six copies gave an 8-fold increase. This activation, which was not dependent on orientation of the TAATGARAT sequence, directly demonstrates that TAATGARAT is a component of the IE gene activation sequence.
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PMID:A modular system for the assay of transcription regulatory signals: the sequence TAATGARAT is required for herpes simplex virus immediate early gene activation. 299 6

We identified an Epstein-Barr virus (EBV) gene product which functions in transient-expression assays as a nonspecific trans activator. In Vero cells, cotransfection of the BglII J DNA fragment of EBV together with recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene gave up to a 100-fold increased expression of CAT activity over that in cells transfected with the recombinant CAT constructs alone. The BglII J fragment acted promiscuously, in that increased CAT synthesis was observed regardless of whether the promoter sequences driving the CAT gene were of EBV, simian virus 40, adenovirus, or herpes simplex virus origin. Cleavage of cloned BglII-J plasmid DNA before transfection revealed that activation was dependent upon the presence of an intact BMLF1 open reading frame. This was confirmed with subclones of BglII-J and with hybrid promoter-open reading frame constructs. This region of the genome is also present in the rearranged P3HR-1-defective DNA species, and defective DNA clones containing these sequences produced a similar activation of CAT expression in cotransfection experiments. The heterogeneous 45-60-kilodalton polypeptide product of BMLF1 may play an important regulatory role in expression of lytic-cycle proteins in EBV-infected lymphocytes.
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PMID:Promiscuous trans activation of gene expression by an Epstein-Barr virus-encoded early nuclear protein. 301 81

To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate-early genes resulted in a marginal increase in ptkCAT induction. This induction was enhanced when the gene for ICP4 was inactivated by restriction enzyme cleavage, substantiating the inhibitory effect of the tsB32 form of ICP4. The two mutant ICP4 genes (tsB32 and tsL14) were unable to trans-activate either of the late CAT constructs (p5CAT and pL42CAT) tested. Cotransfecting tsL14 ICP4 with the other immediate-early genes resulted in activation of p5CAT but not pL42CAT. Taken together, these studies demonstrate that (i) low levels of wild-type ICP4 have stimulatory effect on immediate-early promoters and that higher concentrations of wild-type ICP4 have an inhibitory effect on these promoters, (ii) isolated mutant form of ICP4 exhibit activities that reflect the phenotypes of the mutants from which they were isolated, and (iii) immediate-early gene products other than ICP4 are involved in determining the distinct phenotypes of the two mutants at 39 degrees Celsius.
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PMID:Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4. 301 43

To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT). Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme. Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence. The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles. Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro. Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor. This is in contrast to results found in prokaryotic systems. These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing.
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PMID:The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes. 302 97

Transcription of the vasoactive intestinal polypeptide (VIP) gene is regulated by cAMP. To identify the nucleotide sequences in the human VIP gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5'-flanking region of the human VIP gene fused to the structural sequence encoding the bacterial reporter enzyme chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat pheochromocytoma cell line PC12 were assayed by measuring CAT activity in the cell lysates. Forskolin, an adenylate cyclase-activating agent, stimulated the expression of VIP-CAT fusion genes. Deletional analysis demonstrated that a region between -86 and -70 nucleotides upstream from the transcriptional origin of the human VIP gene was responsible for stimulation by forskolin. This region was able to confer cAMP-responsiveness to a gene that is not normally regulated by cAMP. Two copies of a 5 base pair motif, 5'-CGTCA-3', are required for activity of the VIP cAMP regulatory region. This motif is also present in the cAMP regulatory region of several other eukaryotic genes.
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PMID:Identification of a region in the human vasoactive intestinal polypeptide gene responsible for regulation by cyclic AMP. 303 25

The glucagon gene is expressed specifically in the alpha cells of the pancreatic islets. We show here that 300 base pairs of the 5'-flanking region of the rat glucagon gene, linked to a chloramphenicol acetyltransferase reporter plasmid transfected into islet cell lines of different hormone-producing phenotypes, directs transcription only in glucagon-producing islet cells. Deletional and linker-scanning mutations and DNase I footprinting assays identify three transcriptional control elements within these 300 base pairs. Two of these elements (G2 and G3) independently display enhancerlike functions on both homologous and heterologous promoters in glucagon (alpha) cells, but only on heterologous promoters in insulin- (beta) and somatostatin- (delta) expressing cells, and not in non-islet cells. The proximal promoter element (G1), characterized by low intrinsic transcriptional activity, is critical for specific expression of the glucagon gene in alpha cells. However, nuclear extracts prepared from all three islet cell phenotypes give similar protection to the three control elements of the glucagon 5'-flanking sequence. We conclude that these phenotypically distinct islet cell lines all contain regulatory DNA-binding proteins interacting with the three control elements of the glucagon gene, but that factors interacting with the glucagon promoter result in transcriptional activation only in alpha cells, to restrict glucagon gene expression to these cells. These observations suggest that interactions of nuclear proteins with cis-control elements are involved in the programmed developmental expression of the islet polypeptide hormone genes.
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PMID:Alpha-cell-specific expression of the glucagon gene is conferred to the glucagon promoter element by the interactions of DNA-binding proteins. 306 72

The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated. Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A. Together, these deletions span the region from amino acid 15 to the end of the protein. None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide. These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987). The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase.
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PMID:Extracellular release of colicin A is non-specific. 331 27

The human metallothionein (MT)-IG gene (hMT-IG) is tandemly linked in a head-to-head fashion with the hMT-IF gene. The hMT-IG gene encodes a MT-I polypeptide and has a tripartite structure. The 5'-flanking region of the hMT-IG gene has a TATAA box, four GC motifs, and at least four metal responsive elements. The 3'-untranslated region has a variation of the polyadenylation signal, AATTAA, and the 3'-flanking region a YGTGTTYY RNA processing signal. This gene is expressed in hepatoma-derived cell lines (Hep G2 and Hep3B2) in response to the heavy metals (cadmium, copper, and zinc) but not to the glucocorticoid analogue dexamethasone. In contrast, the lymphoblastoid cell line (Wi-L2) does not express the hMT-IG gene. These results suggest that the hMT-IG gene is regulated differentially and in a cell type-specific manner. Transient expression studies of the chloramphenicol acetyltransferase (CAT) gene under the transcriptional control of either the hMT-IG or hMT-IF promoter in Hep G2 cells has demonstrated that both promoters contain all the necessary cis-acting elements to elicit a similar pattern of heavy metal inducibility. However, the hMT-IG promoter in all instances is five times more active than the hMT-IF promoter. The differences in promoter activity of these genes could possibly be due to inherent differences in their basal level regulatory sequences. The expression of MT-IGcat in transfected Wi-L2 cells demonstrates that the hMT-IG promoter is not cell type-specific.
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PMID:Structure and expression of the human metallothionein-IG gene. Differential promoter activity of two linked metallothionein-I genes in response to heavy metals. 340 43


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