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)

Products of ras oncogenes strongly stimulate the activity of the reporter gene, chloramphenicol acetyltransferase (CAT), driven by a 1.2 kb fragment of the murine cytomegalovirus (MCMV) immediate early (IE) gene enhancer (pCMVCAT). To define the role of proteins binding to the unique cAMP response element (CRE) present in the IE enhancer, NIH 3T3 cells were cotransfected with prasZip6 plasmid, a mammalian expression vector containing a v-Ha-ras cDNA, together with p(delta)ACMVCAT (pCMVCAT without the CRE sequence). Lower stimulation of CAT activity was indeed observed upon deletion of the CRE sequence. Decreased levels of p(delta)ACMVCAT were also observed in cell lines carrying stably transfected ras oncogenes. Further support for the role of the CRE sequence in MCMV enhancer activation comes from the finding that v-Ha-ras expression increases the activity of a reporter gene, beta-galactosidase, driven by three tandem copies of CRE sequence about six-fold. Moreover, this transactivation was prevented by cotransfection of the dominant inhibitor mutant Ha-ras (Leu-61; Ser-186) and was not suppressed by cotransfection of Ha-ras (Asn-17), suggesting that the effect is due to activated ras protein, rather than normal p21ras. Finally the transactivation observed is accompanied by an increase in nuclear proteins binding to a labelled oligonucleotide homologous to the CRE sequence, as shown in a gel retardation assay. These results suggest that the CRE element contributes to the transactivation of the MCMV IE gene enhancer by ras oncogenes.
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PMID:cAMP response element of murine cytomegalovirus immediate early gene enhancer is transactivated by ras oncogene products. 904 20

The ability of zebrafish skeletal muscles to internalize and express plasmid DNA was demonstrated using pCMVCAT1, a chloramphenicol acetyltransferase (CAT) construct driven by the human cytomegalovirus immediate early (CMV-IE) promoter. We found that CAT activity was correlated to the amount of plasmid DNA injected, with maximal expression at 5 micrograms of pCMVCAT1. CAT activity was also shown to increase steadily over the first seven days after injection, with high levels of CAT expression persisting up to one year. Intramuscular injection of CAT constructs driven by other viral promoters also resulted in high levels of CAT activity. Histochemical localization using a CMV beta-galactosidase construct confirmed that only myofibers at the site of injection expressed beta-galactosidase enzyme. The persistence and strong expression of injected plasmid constructs suggest that zebrafish may be a simple and readily accessible system for direct muscle injection studies.
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PMID:Efficient gene transfer into zebrafish skeletal muscle by intramuscular injection of plasmid DNA. 920 Aug 36

For the application of gene therapy to bladder cancer, we examined four in vivo gene transfer methods without viral vectors. For lipofection cationic liposomes (Lipofectin) were instilled into murine bladders. The hemagglutinating virus of Japan (HVJ)-liposomes possessing membrane fusion activity were also injected intraluminally. Using a particle gun, rabbit bladder mucosa was bombarded with DNA-coated gold microcarriers. Electrotransfection was examined in rabbit bladder by pulse direct currents (0.15-0.2 A, 50 msec, repeated 8 times) generated between needle electrodes after submucous injection of DNA solution. beta-galactosidase gene and chloramphenicol acetyltransferase (CAT) gene were used as marker genes. Although lipofection was inefficient in normal urothelium, cancerous urothelium was transfected slightly. HVJ-liposomes more efficiently transfected superficial layers of urothelium with a peak of expression on day 5. The particle gun produced non-uniform but efficient transfection in deeper layers of the urothelium. By electrotransfection, submucous interstitial cells were transfected as well as urothelium. No major complications were observed after these four procedures. HVJ-liposomes are potentially useful for the treatment of carcinoma in situ and the latter two methods may be suitable for the adjuvant therapy of localized bladder tumors.
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PMID:[In vivo gene transfer methods into bladder without viral vectors]. 943 30

The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells. The level of Cre expression is critical to induce loxP site-specific recombination in ES cells. To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (CAG) promoter, human polypeptide chain elongation factor 1alpha (hEF-1alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct. Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression. No selection system was used in the experiments. The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1alpha promoter, the mPGK promoter and the MC1 promoter in order. These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector. Thus, it is important to choose the promoter for effective recombination by Cre.
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PMID:Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters. 944 13

Amino acid (aa) mutations in the interferon-sensitivity determining region (ISDR) (aa position 237-276 of the nonstructural region 5A [NS5A] protein consisting of 447 amino acids) of hepatitis C virus (HCV) are related to increased interferon sensitivity and low viral load, but its mechanism has not been clarified. Recently, the NS5A protein has been reported to have a transcriptional activation function, like other viral transactivator proteins known to repress interferon-induced gene expression, and the ISDR overlaps one of the acidic amino acid regions, putative domains conferring this activity. In the present study, we investigated the transcriptional activation function of the ISDR itself and the effect of amino acid mutations in the ISDR on this activity. The full-length or truncated NS5A cDNA with different ISDR sequences was cloned into a yeast or mammalian expression vector to form a fusion protein consisting of the GAL4 DNA-binding domain (GAL4-DBD) and NS5A protein. Following transfection, the transcriptional activities of these constructs were determined using beta-galactosidase (yeast) or chloramphenicol acetyltransferase (CAT) (mammalian cell) reporter gene expression under the control of GAL4 binding sites. In yeast, both the full-length sequence of NS5A-R (a clone with one aa mutation in the ISDR) and NS5A-S (a derivative of NS5A-R with six aa mutations in the ISDR) had no distinguishable transcriptional activity, whereas an amino-terminal deletion construct of NS5A-R (aa position 228-447) lacking 227 aa, showed remarkable activity with the relative value of 117.0 over that of the backbone vector. The same deletion mutant of NS5A-S produced five times higher activity with the relative value of 575.0, indicating that aa mutations in the ISDR profoundly affect this transcriptional activity. In a hepatoma cell line, HuH-7, the transcriptional activity was more prominent with a construct consisting of only the ISDR and short flanking sequences (aa 228-284) than larger deletion constructs of NS5A-R. Analysis using six different ISDR clones revealed that different mutations enhanced this activity to various extent compared with the wild-type ISDR. In particular, site-directed mutagenesis targeted to the aa position 252 showed that this aa residue had profound influence on the activity. These results suggest that the ISDR has a transcriptional activity, and it is enhanced by aa mutations that are also related to decreased viral load and increased interferon sensitivity. The possible association between transcriptional activation and interferon sensitivity or viral replication should be studied further.
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PMID:Mutations in the interferon-sensitivity determining region of hepatitis C virus and transcriptional activity of the nonstructural region 5A protein. 975 55

The "Fab immunogene" is a novel gene transfer vehicle in which the Fab fragment of anti-human epidermal growth factor (EGF) receptor antibody B4G7 is conjugated with poly-L-lysine to form an affinity complex with DNA. It was developed to target delivery of therapeutic genes into EGF receptor-hyperproducing tumor cells. Various characteristic features of the immunogene have been documented (Chen et al., 1998). Here we add further evidence to prove that in vitro transfer of beta-galactosidase/Fab immunogene is exclusively to EGF receptor-positive cells and that the herpes simplex virus thymidine kinase (TK)/Fab immunogene induces substantial suicide effects on A431 tumor cells when treated together with ganciclovir. The in vivo specificity of the immunogene transfer was examined using A431 tumor-bearing nude mice. When these nude mice were injected intraperitoneally with the chloramphenicol acetyltransferase (CAT)/Fab immunogene, CAT DNA was detected in the tumors as well as in liver and kidney but not brain, whereas CAT mRNA and enzyme activity were detected only in the tumors. Local and intraperitoneal injection of the TK/Fab immunogene and subsequent administration of ganciclovir effectively suppressed the growth of A431 tumors transplanted on the backs of nude mice. These observations suggest a possible application of the Fab immunogene system in cancer gene therapy.
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PMID:Targeted in vivo delivery of therapeutic gene into experimental squamous cell carcinomas using anti-epidermal growth factor receptor antibody: immunogene approach. 987 65

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.
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PMID:Transcriptional regulation of the Bmp2 gene. Retinoic acid induction in F9 embryonal carcinoma cells and Saccharomyces cerevisiae. 988 May 12

Cellular nuclease activity is a potential barrier to the successful delivery of foreign genes to mammalian cells. We tested the hypothesis that transfection in the presence of a specific DNase inhibitor can enhance the expression of foreign gene products. We have used DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously to inhibit porcine DNase II, an acid pH nuclease contained in the endosomal/lysosomal compartment. Transfection of H441 cells in the presence of 0.1-1 microgram/ml DMI-2 caused: (1) 10-fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5- to two-fold enhancement of CAT activity in cells exposed to lipofectin-DNA complexes: (3) no effect on transfection via calcium phosphate co-precipitation. DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly-lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 increased the number of transfected cells. Methanolysis products of DMI-2 did not inhibit DNase II and did not enhance transfection efficiency. Taken together, the data support the hypothesis that nuclease action is a significant barrier to expression of foreign genes and inhibition of specific nucleases may facilitate transfection.
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PMID:Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor. 993 Mar 26

Noncytopathic replicons of the flavivirus Kunjin (KUN) were employed for expression and delivery of heterologous genes. Replicon vector C20DX2Arep, containing a unique cloning site followed by the sequence of 2A autoprotease of foot-and-mouth disease virus, was constructed and used for expression of a number of heterologous genes including chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), beta-galactosidase, glycoprotein G of vesicular stomatitis virus, and the Core and NS3 genes of hepatitis C virus. The expression and proper processing of these genes upon transfection of BHK21 cells with the recombinant replicon RNAs were demonstrated by immunofluorescence, radioimmunoprecipitation, and appropriate reporter gene assays. Most of these recombinant KUN replicon RNAs were also successfully packaged into secreted virus-like particles (VLPs) by subsequent transfection with Semliki Forest virus replicon RNA expressing KUN structural genes. Infection of BHK21 and Vero cells with these VLPs resulted in continuous replication of the recombinant replicon RNAs and prolonged expression of the cloned genes without any cytopathic effect. We also developed a replicon vector for generation of stable cell lines continuously expressing heterologous genes by inserting an encephalomyelocarditis virus internal ribosomal entry site-neomycin transferase gene cassette into the 3'-untranslated region of the C20DX2Arep vector. Using this vector (C20DX2ArepNeo), stable BHK cell lines persistently expressing GFP and CAT genes for up to 17 passages were established. Thus noncytopathic KUN replicon vectors with the ability to be packaged into VLPs should provide a useful tool for the development of noninfectious and noncytopathic vaccines as well as for gene therapy applications.
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PMID:Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes. 1006 62

p73 is a recently cloned tumor suppressor gene that is highly homologous to p53, and the products of both possess similar functions in inhibiting cell growth and inducing apoptosis. Interestingly, the COOH-terminal region of p53 displays no significant homology with that of p73. Moreover, p73 has an additional segment at its COOH terminus. Recently, we have found two mutations of p73 with amino acid substitution (P405R and P425L) in primary neuroblastomas. Because the region (amino acid residues 382-491) contains a glutamine- and proline-rich domain, we hypothesized that it has a transactivation function, and the mutations found in tumors result in loss of function. To test it, we used the yeast GAL4 DNA-binding fusion system. Yeast transformants expressing a GAL4-p73(1-112) or a GAL4-p73alpha(380-513) fusion protein were grown in SD medium lacking histidine and tryptophan and exhibited a significant induction of beta-galactosidase activity. Transient transfection experiments revealed that both of fusion proteins could induce the chloramphenicol acetyltransferase activity in mammalian cells, indicating that the COOH-terminal as well as NH2-terminal regions of p73 had significantly high levels of transactivation activity. Furthermore, the former activity was severely impaired in two naturally occurring mutant forms found in neuroblastomas. These suggest that, unlike p53, p73 has two domains with transactivation function, one in the NH2-terminal region and the other in the COOH-terminal region. Loss of function mutation in the latter might be involved in tumorigenesis and/or tumor progression.
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PMID:Identification of a transactivation activity in the COOH-terminal region of p73 which is impaired in the naturally occurring mutants found in human neuroblastomas. 1038 37


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