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)

Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy.
Proc Natl Acad Sci U S A 1990 Dec
PMID:In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. 217 6

Stromelysin is a member of the metalloproteinase family which plays an important role in extracellular matrix remodelling during many normal and disease processes. We show here that in polyomavirus-transformed rat embryo fibroblast cells (PyT21), the transcription from the stromelysin gene is repressed by the vitamin A derivative retinoic acid (RA). Furthermore, expression vectors encoding the human RA receptors hRAR-alpha, hRAR-beta and hRAR-gamma repress chloramphenicol acetyltransferase (CAT) expression from stromelysin promoter-CAT gene expression vectors in RA-treated PyT21 and human HeLa cells, as determined by transient transfection assays. Through mutation and deletion analysis, we show that the RA dependent repression is mediated by a 25 bp region from nucleotide positions -72 to -48 of the rat stromelysin 5'-flanking DNA sequence. Further mutation analysis of this region indicates that the DNA sequence required for RA dependent repression colocalizes with an AP1 binding site which is essential for promoter activity. We show also that RA represses the transcriptional activity of a reporter gene containing a TPA responding AP1 binding site driving the HSV tk promoter. Thus the RAR-RA complex appears to repress transcription of the stromelysin gene by blocking activation by positive regulatory factors. However, we found no evidence supporting the possibility that the RA dependent repression could be due to RAR binding to the AP1 binding site or to the AP1 components c-fos and c-jun.
EMBO J 1990 Dec
PMID:Negative regulation of the rat stromelysin gene promoter by retinoic acid is mediated by an AP1 binding site. 217 52

We compared the ability of HIV-1 tat protein and JCV T-antigen in inducing transcription from the JCV late promoter, JCVL. A JCVL promoter-chloramphenicol acetyltransferase plasmid (pJCL-CAT) was transfected into human glial cells alone or together with plasmids producing T-antigen and tat protein. CAT enzyme activity obtained from the transfected cells indicated that both JCV T-antigen and HIV-1 tat proteins stimulated JCV late gene expression. However, the level of induction mediated by tat protein was significantly higher than that obtained with T-antigen. Moreover, in contrast to JCV T-antigen, tat stimulated JCVL-promoter activity over a narrow range of ptat expressor plasmid concentration. Co-transfection of both T-antigen and tat plasmids at optimal concentrations resulted in greater than additive CAT activity from the JCVL promoter. This synergism suggests that the two activator proteins utilize alternative mechanisms to exert their effects. Using deletion mutations from the 5' end of the JCVL promoter, we demonstrated that different regions within the JCV enhancer/promoter are important for T-antigen and tat induction, implying that these activators function through distinct targets to increase JCVL promoter activity.
Oncogene 1990 Dec
PMID:Regulation of the human neurotropic virus promoter by JCV-T antigen and HIV-1 tat protein. 217 36

We and others have introduced the use of the lac operator-repressor system as a method for providing inducible gene expression for gene transfer experiments in animal cells (M. C.-T. Hu, and N. Davidson, Cell 48:555-566, 1987; J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52:713-722, 1988). To improve the dynamic range of such an inducible system, we have investigated the effects of combining the relief by isopropyl-beta-D-thiogalactoside (IPTG) of negative control by the lac system with positive induction by the natural inducers glucocorticoids and cadmium ion for a system based on the human metallothionein-IIA gene promoter. We used the chloramphenicol acetyltransferase gene as a reporter gene and inserted a lacO sequence into the promoter between the GC box and metal-responsive element 1, between metal-responsive element 1 and the TATA box, or between the TATA box and the transcription start site. Surprisingly, all of these insertions had a significant inhibitory effect on promoter activity even in the absence of repressor. However, with these lacO-containing constructs, the levels of gene expression after induction by glucocorticoid, Cd2+, or both were considerably reduced in cells engineered to express the lac repressor. Derepression by IPTG, coupled with induction by both dexamethasone and Cd2+ ion, then provided a high level of induced expression, i.e., by a factor of approximately 100 over the basal level of expression. However, inserting the lacO sequence well upstream just before the glucocorticoid-responsive element had much smaller effects on expression levels in both repressor-negative and repressor-positive cells. This study describes a new, high-level-inducible promoter system for gene transfer experiments. The observed effects are discussed in terms of current models of the mechanisms by which transcription factors control gene expression.
Mol Cell Biol 1990 Dec
PMID:A combination of derepression of the lac operator-repressor system with positive induction by glucocorticoid and metal ions provides a high-level-inducible gene expression system based on the human metallothionein-IIA promoter. 224 53

A novel cytokine, interleukin-8 (IL-8), may play major roles in the inflammatory process by recruiting neutrophils and T cells into inflammatory sites. The production of this cytokine is not constitutive and is induced in a variety of cell types by stimulation with mitogens and cytokines. Among cytokines, only IL-1 and tumor necrosis factor (TNF) can induce IL-8 gene expression at the transcriptional level. Transfection of a human fibrosarcoma cell line with chloramphenicol acetyltransferase expression plasmids linked to a 5'-flanking deletion mutants of the IL-8 gene demonstrated that the nucleotides between -94 and -71 base pairs from the start of the first exon are essential and sufficient for the IL-8 induction by either IL-1, TNF, or phorbol 12-myristate 13-acetate. This sequence is composed of two cis-elements; one is the potential binding site for a nuclear factor-kappa B-like factor and the other for a cis-regulatory enhancer binding protein-like factor. Mutations in either elements abolished IL-1, TNF, and phorbol 12-myristate 13-acetate responsiveness. This report provides the first evidence that cooperation between two distinct cis-elements may be required for induction of gene expression by either IL-1 or TNF.
J Biol Chem 1990 Dec 05
PMID:Cooperative interaction of nuclear factor-kappa B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. 225 17

Nucleotide sequences that direct transcription of the human 5-lipoxygenase gene have been examined by ligation to the chloramphenicol acetyltransferase gene and determination of chloramphenicol acetyltransferase activity in transfected HeLa and HL-60 cells. Various lengths of 5'-flanking sequences up to 5.9 kilobase pairs 5' of the transcriptional initiation sites were tested. Two positive and two negative apparent regulatory regions were seen. Part of the promoter sequence (-179 to -56 from ATG), which includes five repeated GC boxes (the putative Sp1 binding sequence) was essential for transcription in both HeLa and HL-60 cells. Gel-shift assays (using the DNA fragment -212 to -88) revealed that the transcriptional factor Sp1 could bind to this region of the 5-lipoxygenase promoter. Furthermore, HL-60 nuclear extracts contained specific nuclear factor(s) binding to 5-lipoxygenase promoter DNA, which could not be detected in HeLa cell nuclear extracts.
Proc Natl Acad Sci U S A 1990 Dec
PMID:Characterization of the human 5-lipoxygenase gene promoter. 225 Dec 50

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express beta-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete beta-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine beta-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency (greater than 150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the beta-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous beta-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.
Proc Natl Acad Sci U S A 1990 Dec
PMID:Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells. 225 Dec 52

Genomic and cDNA clones encoding the murine pulmonary surfactant protein SP-C were isolated and sequenced to provide the primary amino acid sequence of the murine SP-C polypeptide and the structural organization of the SP-C gene locus. Murine SP-C is encoded by a single locus spanning approximately 3.2 kilobases of genomic DNA. The gene is composed of six exons and five introns, and its structure is closely related to the human SP-C gene. The primary transcript for the murine SP-C preprotein of Mr = 21,000 has an overall nucleotide identity of 80% with the coding sequence of the human precursor. The active airway peptide of 33-35 amino acids was even more closely related to the human SP-C airway peptide, sharing 95% identity at the amino acid level. The most significant structural difference between the murine and human SP-C genes was an increased size of the first intron in the murine SP-C gene. A single mRNA encoding murine SP-C of 0.8 kilobase was detected only in lung tissue, and SP-C mRNA was more abundant in neonatal and postnatal lung tissue than in fetal lung tissue. The nucleotide sequence of the promoter and 5'-flanking regions of the murine SP-C gene shared considerable identity with the human SP-C gene. Transgenic mice bearing a chimeric gene composed of the 5'-flanking and promoter regions of the human SP-C gene and the bacterial chloramphenicol acetyltransferase reporter gene were generated, demonstrating that lung-specific and developmental expression was conferred by these 5'-sequences. The chimeric gene was selectively expressed in preparations of respiratory epithelial cells, but was not expressed in alveolar macrophages isolated from the transgenic mice. The shared structural organization and developmental and tissue-specific expression of the SP-C gene between the mouse and human demonstrate considerable phylogenetic conservation of this gene and protein.
J Biol Chem 1990 Dec 15
PMID:Structure and expression of the pulmonary surfactant protein SP-C gene in the mouse. 225 41

The murine gene for adipocyte P2 encodes an adipocyte-specific member of the family of intracellular lipid binding proteins. The region upstream from the start of transcription of this gene has been found to contain binding sites for the transcription factors c-jun/c-fos and C/EBP (CCAAT/enhancer binding protein) and several short sequence elements found in other adipocyte gene promoters, termed fat-specific elements. To identify DNA sequences that were responsible for the high level of transcription of the gene for adipocyte P2 in vivo, we made a series of transgenic mice containing 168 base pairs (bp), 247 bp, 1.7 kilobases (kb), and 5.4 kb of 5' flanking sequence linked to the bacterial gene chloramphenicol acetyltransferase. Although plasmids containing only 168 bp of 5' sequence including the C/EBP and AP-1 (activation protein 1) binding sites were expressed well in cultured adipocytes, high levels of chloramphenicol acetyltransferase activity in the adipose tissue of transgenic mice were not observed until the 5' flanking region was extended to kb -54. An enhancer mapping between kb -4.9 and kb -5.4 upstream from the start of transcription was identified by transfection of further deletions into cultured adipocytes. This enhancer, when linked to a bp -63 promoter fragment from the gene for adipocyte P2, directed very high level chloramphenicol acetyltransferase expression specifically to adipose tissue in transgenic mice. These results identify a functional adipose-specific enhancer and indicate that it is the major determinant of tissue specificity of the gene for adipocyte P2. These results also demonstrate that the proximal-promoter binding sites for AP-1 and C/EBP are not sufficient or necessary to give adipose-tissue-specific expression in vivo, though they may play an important role in the response of this promoter to glucocorticoids.
Proc Natl Acad Sci U S A 1990 Dec
PMID:A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2 in vivo. 226 14

1. The type III variant of chloramphenicol acetyltransferase (CATIII) is resistant to inactivation by ionizable modifying reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and iodoacetate, whereas it is sensitive to inhibition by similar but uncharged reagents, including 4,4'-dithiodipyridine, methyl methanethiolsulphonate (MMTS) and iodoacetamide. The target for these thiol-modifying reagents has been postulated to be Cys-31. This residue is situated within a part of the chloramphenicol-binding site formed largely from the side chains of hydrophobic amino acid residues, which might be expected to discriminate against the access of ionized ligands to Cys-31. 2. The substitution of Cys-31 by alanine, serine, threonine or methionine yields an enzyme that is resistant to inactivation by thiol-specific reagents. Replacement of Cys-31 by alanine, serine or threonine results in increased Km values for chloramphenicol with only small changes in kcat.. In contrast, the Cys-31----Met substitution mainly affects kcat. values. Although the kcat. for chloramphenicol acetylation is decreased 13-fold compared with wild-type CAT, the kcat. for the acetyl-CoA hydrolysis reaction, which occurs in the absence of chloramphenicol, is increased 2.7-fold. 3. MMTS modification of cysteine residues results in an adduct (-CH2-S-S-CH3) that is structurally similar to the side chain of a methionine residue (-CH2-CH2-S-CH3). The kinetic properties of MMTS-modified CATIII closely resemble those of [Met31]CAT.
Biochem J 1990 Dec 01
PMID:Elimination of a reactive thiol group from the active site of chloramphenicol acetyltransferase. 226 77


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