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)

We have constructed a series of replication-competent retrovirus vectors to introduce and express gene cassettes in avian cells. To characterize these vectors, we inserted the coding sequences for the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the chicken beta-actin gene promoter or the mouse metallothionein 1 gene promoter. In all cases, we found the structure of integrated proviruses to be stable during serial cell passage in vitro. Chloramphenicol acetyltransferase activity was detected biochemically and immunocytochemically in infected cells. Cassettes were inserted in the vectors in the same or in the opposite orientation with respect to viral transcription. Although both orientations were functional, the cassettes inserted in the forward orientation were usually expressed at higher levels than the corresponding backward constructions. The level of expression was strongly influenced by surrounding proviral sequences, particularly by the transcriptional enhancer elements within the retrovirus long terminal repeat sequences. Expression was higher with vectors that contained the polymerase (pol) region of the Bryan high-titer strain of Rous sarcoma virus. Inclusion of the Bryan pol region also improved vector replication in the chemically transformed quail fibroblast line QT6.
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PMID:Replication-competent retrovirus vectors for the transfer and expression of gene cassettes in avian cells. 204 Oct 92

Two alternating purine-pyrimidine sequences of the d(TG)n.d(CA)n-type (170bp and 60 bp in length) lie upstream of the rat prolactin (rPRL) gene. Conformational studies of plasmids containing these sequences indicate that both form left-handed (Z) DNA, with transitions initiating at superhelical densities of -0.041 and -0.044 respectively. These alternating purine-pyrimidine (APP) sequences are hypersensitive to cleavage with S1 nuclease both at the boundaries and within these APP repeats, where there is a loss in APP alternation. We have investigated the function of one of these Z-DNA sequences in the regulation of rPRL transcription, by linking regions of the 5' flanking sequence of the rPRL gene to a reporter gene encoding chloramphenicol acetyltransferase (CAT), and transferring these plasmids into GH3 pituitary tumour cell lines. The major conclusion from these studies is that the 170bp repeat exerts a negative effect on the transcription of the rPRL gene, and also down-regulates the expression of the fusion gene pRSVcat when cloned 50bp upstream of the Rous sarcoma virus promoter. However, despite its proximity to an estrogen response element in prolactin, this sequence does not affect the responsiveness of the rPRL gene to estrogen.
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PMID:d(TG)n.d(CA)n sequences upstream of the rat prolactin gene form Z-DNA and inhibit gene transcription. 215 81

Dexamethasone treatment of the Jurkat T77 cell clone inhibited the enhancing effect of 12-O-tetradecanoylporbol-13-acetate (TPA) and the calcium ionophore A23187 on the interleukin 2 (IL2) mRNA levels and gene transcription from intact nuclei. Dexamethasone treatment of Jurkat T77 cells inhibited the TPA/A23187-dependent activation of the transcription from the transfected pIL2CAT, containing 600 base pairs of the genomic sequences upstream of the coding region of IL2 gene, including the TPA/calcium responsive cis-regulatory elements and promoter sequences, driving the expression of the chloramphenicol acetyltransferase (CAT) gene. Transfection of either Jurkat T77 cell clone or glucocorticoid-resistant Jurkat cells with a human glucocorticoid receptor cDNA under the transcriptional control of the Rous sarcoma virus long terminal repeat (LTR) (pRShGR alpha) significantly increased or induced, respectively, the dexamethasone-mediated inhibition of the TPA/A23187-dependent expression of pIL2-CAT as well as the enhancing effect on the expression of the cotransfected CAT gene under the control of the mouse mammary tumor virus LTR, as a marker of glucocorticoid receptor action. This suggests a role for the glucocorticoid receptor in mediating the dexamethasone action on IL2 gene expression. To study the cis-regulatory sequence specificity of the dexamethasone-induced interference with the TPA/A23187-mediated T cell activating signals, we studied the effect of the hormone on the regulatory elements contained in the Rous sarcoma virus and human T lymphotropic virus 1 long terminal repeats and the SV40 promoter, which are known to be transcriptionally enhanced by those activating agents. Dexamethasone was unable to interfere with the TPA/A23187-mediated enhancement of these cis-regulatory elements, suggesting that the hormone effect is specific for IL-2 gene sequences. Our data suggest that the dexamethasone-mediated transcriptional inhibition of the IL2 gene is mediated by an interference with the protein kinase C and calcium-mediated trans-activation of the antigen-responsive and T cell-specific elements lying in the 5'-flanking region of the gene.
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PMID:Transcriptional regulation of the interleukin 2 gene by glucocorticoid hormones. Role of steroid receptor and antigen-responsive 5'-flanking sequences. 215 67

Transient expression of chloramphenicol acetyltransferase (CAT) was used to study Marek's diseases virus (MDV)-mediated transactivation of the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter. Cotransfection experiments in primary avian cells were conducted using MDV high-molecular-weight DNA and plasmid pRSVcat. Increased CAT activity, relative to controls, was consistently observed in the presence of MDV. Enhanced CAT activity, expressed via the RSV-LTR promoter, was strictly dependent on the presence of MDV DNA or virus, suggesting that activation of the RSV-LTR promoter was due to factors expressed in MDV-infected cells. Differences in transactivation efficiency were observed between various strains and the serotypes of MDV. In particular, high- and low-passage pairs of serotype 1 MDV showed marked differences in their ability to increase CAT activity in pRSVcat-transfected cells. Attenuation of viral pathogenicity and decreased expression of some cell surface glycoproteins occur in high-passage MDV strains. Decreased transactivation ability in these same strains suggests that continuous passage in culture and attenuation may perturb a regulatory mechanism operating by transcriptional control. In addition, transactivation of the RSV-LTR promoter suggests that increased incidence of avian leukosis following vaccination by MDV may be due to MDV-mediated transactivation of endogenous ALV proviral LTR promoters. MDV-mediated transactivation was not limited to the RSV-LTR promoter. Serotype 3 MDV (HVT) efficiently transactivated the herpes simplex virus (HSV) alpha 4 (ICP4) and beta-TK promoters as well as the human cytomegalovirus (hCMV) immediate early promoter.
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PMID:Transactivation of the Rous sarcoma virus long terminal repeat promoter by Marek's disease virus. 217 59

The gene encoding dihydrofolate reductase (DHFR) is down-regulated as myoblasts withdraw from the cell cycle and commit to terminal differentiation. To localize cis-acting elements involved in regulating DHFR gene expression, the DHFR promoter and upstream region, together with differing amounts of contiguous intragenic sequence, were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The resulting fusion genes were stably transformed into muscle cells, and CAT mRNA levels were measured in proliferative myoblasts and committed myocytes. A gene consisting of the -850/+465 region of DHFR (numbers refer to distance in base pairs from transcription initiation site) fused to CAT was efficiently expressed in proliferating myoblasts and was appropriately down-regulated during commitment. A gene consisting of the -850/+60 region of DHFR fused to CAT was poorly expressed in proliferating myoblasts and was not down-regulated during commitment. When inserted between the Rous sarcoma virus promoter and CAT sequence of RSVpCAT, the +61/+465 region of the DHFR gene augmented CAT mRNA expression in muscle cell transformants but did not confer a regulated pattern of expression. Our data indicate that DHFR sequences between +60 and +465 are required but are not sufficient for replication-dependent expression. The DHFR sequences may be operating at either a transcriptional or posttranscriptional level.
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PMID:An intragenic region downstream from the dihydrofolate reductase promoter is required for replication-dependent expression. 221 31

Activities of heterologous promoters and enhancers in cultured rainbow trout liver cells were examined employing the bacterial chloramphenicol acetyltransferase gene as the reporter. SV40 promoter-enhancer and Rous sarcoma virus long terminal repeat directed constitutive expression at high levels. Moloney murine leukemia virus long terminal repeat and SV40 promoter combined with Adenovirus type 2 enhancer were also constitutively expressed. Drosophila Hsp70 promoter was activated when the transformed cells were cultured at 25 degrees C, a higher temperature than the temperature normally used, in faithful response to heat shock.
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PMID:Constitutive and inducible expression of a transgene directed by heterologous promoters in a trout liver cell line. 226 33

The small marine ostracod crustacean, Vargula hilgendorfii, produces a bright blue luminous secretion which is ejected into seawater. The luminescence is due to a simple enzyme-catalyzed reaction involving only luciferase, luciferin (substrate), and molecular oxygen. Thus, V. hilgendorfii luciferase (VL) should be useful as a reporter enzyme in studies of gene expression in mammalian cells. Expression plasmids consisting of VL cDNA (vl) linked to the promoters simian virus 40 early region, Rous sarcoma virus long terminal repeat, human elongation factor, or mouse granulocyte colony-stimulating factor were introduced into a series of mammalian cell lines. Following transfection, VL activities in cell extracts and culture media were determined by a rapid light emission assay with V. hilgendorfii luciferin. Parallel experiments were carried out with the chloramphenicol acetyltransferase (CAT)-encoding gene. In all cell lines tested, VL was secreted, allowing the reporter activity to be determined directly from a small aliquot of the culture medium. The results indicate that the secreted VL enzyme is superior to CAT, firefly luciferase, and bacterial luciferase as a convenient and versatile indicator of gene expression in mammalian cells.
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PMID:Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells. 226 35

To study the frequency of germ-line transformation and to examine the reproducibility of tissue-specific transgene expression, we produced several lines of transgenic zebrafish expressing a recombinant chloramphenicol acetyltransferase (CAT) gene. Supercoiled plasmids containing both Rous sarcoma virus and SV-40 promoter sequences upstream of the CAT coding region were injected into zebrafish embryos prior to first cleavage. CAT activity could be detected in batches of injected embryos as early as 8 h and up to at least 12 days post-fertilization. Approximately 18% of injected fish raised to maturity exhibited CAT activity in their fins, and approximately 5% of injected fish became stable germ-line transformants. Breeding studies indicated that although transgenic founder fish were frequently germ-line mosaics, transgenic individuals of subsequent generations were fully hemizygous for the transgene marker. The transgenes present in the F1 progeny of four independent lines were relatively well expressed in fin and skin, while lower levels of expression were observed in heart, gill and muscle. Little or no CAT expression was observed in the brain, liver and gonad. A monoclonal antibody directed against the CAT gene product consistently revealed variegated patterns of CAT expression in ectodermally derived fin epidermal cells in three of these lines. These results show that it is possible to efficiently produce stable germ-line transformants of the zebrafish and to observe reproducible tissue-specific patterns of transgene expression in this organism. Possible mechanisms for the variegated expression observed within tissues are also considered.
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PMID:Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression. 240 Dec 11

To distinguish the inhibitory effect of anti-sense RNA on translation from the effect on splicing, a plasmid (pLC32) was constructed from a cDNA clone of the Rous sarcoma virus (RSV) envelope gene (env) mRNA. Transcription of this plasmid results in the synthesis of RNA identical to the RSV env gene mRNA which does not require splicing to be expressed. Plasmids derived from pLC32 were also constructed in which the env gene coding sequence and 5' noncoding leader sequences were inserted in the opposite orientation relative to the RSV long terminal repeats (LTRs). pLC32 DNA transfected by the calcium phosphate coprecipitation technique efficiently rescued infectious virus from quail cells infected with an RSV mutant deleted in the env gene [R(-)Q cells], indicating that the intron sequences are dispensable in env gene expression. When the inverted constructs were cotransfected with pLC32, significantly less infectious virus was produced. The extent of the inhibition depended upon the concentration ratio of the two plasmids. The maximum inhibition (80%) occurred when the ratio of inverted constructs to pLC32 was 12:1. The inhibition is specific for the inverted orientation since cotransfection of pLC32 with several other plasmids containing viral LTRs and defective src and env genes at similar concentrations did not inhibit the production of infectious virus. In addition, the inverted constructs did not interfere with the expression of an LTR-driven chloramphenicol acetyltransferase gene. When cotransfected with a wild-type Prague A RSV DNA plasmid (pJD100), the inverted constructs also greatly inhibited expression and replication of virus in R(-)Q quail cells. These data suggest that the specific inhibition is caused by hybridization of complementary RNA transcribed from the inverted constructs to the env mRNA, thereby blocking its expression. The fact that expression of both intron-containing and intronless clones are inhibited to the same extent suggest that inhibition by anti-sense RNA from the env exon regions does not act at the level of RNA splicing.
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PMID:Gene expression from both intronless and intron-containing Rous sarcoma virus clones is specifically inhibited by anti-sense RNA. 242 79

We have constructed nonpermuted replication-competent avian retrovirus vectors that derive from Rous sarcoma virus (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We describe here the construction and properties of corresponding vectors in which the long terminal repeats (LTRs) of the parental virus have been replaced by the LTRs of the endogenous chicken virus Rous-associated virus type O. The Rous-associated virus type O LTR vectors replicated approximately 1/10 as well as the parental vectors and expressed a test gene, chloramphenicol acetyltransferase, approximately 1/30 to 1/50 as well.
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PMID:Helper-independent retrovirus vectors with Rous-associated virus type O long terminal repeats. 246 Jun 45


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