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)

Complementary DNA (cDNA) clones encoding human-androgen receptors (haR) were isolated using synthetic oligonucleotides homologous to the human glucocorticoid, estradiol, progesterone, and aldosterone receptors as probes to screen a human testis lambda gt11 cDNA library. One of the receptor proteins (hARa) produced in vitro bound the [3H]dehydrotestosterone ([3H]DHT) with high affinity and selectivity similar to the human androgen receptor present in target tissues and cells. A second cDNA clone (hARb) encoding an identical amino terminal and DNA binding domains, but differing by four amino acids at the hormone binding domain, did not bind [3H]DHT with high affinity when incubated with protein expressed by in vitro transcription-translation. Cotransfection of hARa in an expression vector with mouse mammary tumor virus (MMTV)-bacterial chloramphenicol acetyltransferase chimeric plasmids, followed a hormone-dependent trans-activation, defining the binding affinity of hARa between 5 x 10(-10) and 1 x 10(-9) M for [3H]DHT. A similar cotransfection experiment with hARb indicated a KD of hARb for [3H]DHT to be above approximately 10(-8) M. The deduced primary structures of hARa and hARb contain the viral erbA homologous region found in other steroid, thyroid, and vitamin receptors and is identical to the hAR sequences reported by others. The amino acid sequence differs at the Gly stretch (16 Gly instead of 27, 24 or 23) of the N-terminal domain and in hARb, the sequence reads I.F.F.F.F.L.L (816-822) instead of K.F.F.D.E-L (816-821) in the hARa and other reported hAR sequences. The difference of four amino acids in the steroid binding domain of hARb is associated with altered DHT binding and thus a lack of trans-activation by way of AR responsive elements in MMTV-long terminal repeat. The interaction of hARa and hARb with synthetic responsive elements by gel-retardation assay and their responsiveness in trans-activation by calcium phosphate coprecipitation demonstrates that hARb can inhibit trans-activation by hARa in this system.
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PMID:Specific region in hormone binding domain is essential for hormone binding and trans-activation by human androgen receptor. 234 76

Fast skeletal muscle troponin C (sTnC) is the calcium-binding subunit of the myofibrillar thin filament that regulates excitation-contraction coupling. Utilizing a polymerase chain reaction cloning strategy, we have isolated cDNA clones encoding murine sTnC. The 160-amino acid sTnC protein shares 70% amino acid sequence identity with the slow/cardiac isoform of troponin C (cTnC). However, three areas of significant sequence divergence were identified. Southern blot analyses demonstrated that murine sTnC is encoded by a single copy gene that is distinct from that which encodes cTnC. Northern blot analyses showed that the sTnC gene is expressed exclusively in skeletal muscle (extensor digitorum and anterior tibialis) and not in neonatal or adult heart, brain, kidney, liver, lung, or testes. Studies of the murine C2C12 myoblast cell line demonstrated that sTnC gene expression is developmentally regulated during the differentiation of these myoblasts into myotubes. A full-length murine sTnC genomic clone was isolated and characterized by DNA sequence, primer extension, and S1 nuclease protection analyses. The sTnC gene is composed of six exons spanning 2.6 kilobase pairs of genomic DNA. Although the introns do not divide the gene into functional domains, the intron-exon borders are nearly identical to those of the other members of the troponin C multigene family. Transient transfection assays using chloramphenicol acetyltransferase reporter plasmids demonstrated that the sTnC promoter alone is relatively inactive in muscle cells and that high level sTnC gene expression in these cells is controlled by a potent transcriptional enhancer element located within the first intron of the gene. In additional transfection experiments, the sTnC enhancer was shown to display three important biological activities. (i) It was required for high level transcription from the sTnC promoter in muscle cells; (ii) its activity was muscle cell specific; and (iii) its activity was developmentally regulated during the differentiation of C2C12 myoblasts to myotubes. Taken together, these data define the sTnC gene as an excellent model system for studies of developmentally regulated gene expression in skeletal muscle.
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PMID:The structure and regulation of expression of the murine fast skeletal troponin C gene. Identification of a developmentally regulated, muscle-specific transcriptional enhancer. 239 55

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

Primary murine keratinocytes can be maintained in culture for extended periods in a proliferative, basal cell state under conditions of reduced extracellular Ca2+. In response to increased Ca2+ concentrations, the cells undergo a well-defined program of terminal differentiation, thus serving as a convenient model in which to study the genes involved in regulating this and possibly other differentiation cascades by DNA-mediated gene transfer. However, because of their sensitivity to increased Ca2+ concentrations, the introduction of exogenous genomic DNA into primary keratinocytes by conventional methods is problematic. We have optimized the calcium phosphate DNA transfection procedure by introducing conditions that reduce the potency of Ca2+ as a differentiation signal. Primary epidermal cells were transfected with pSV2CAT, a plasmid that codes for the enzyme chloramphenicol acetyltransferase CAT. Enzyme activity was measured in cell extracts under varying transfection conditions. When the K+ concentration of the medium used for transfection by calcium phosphate precipitation is reduced from 6.5 to 0.01 mM, CAT activity following transfection increases 2-3 times. Exposure to the DNA precipitate for 2-4 h is optimal. By the use of fibroblast conditioned medium following transfection, enzyme activity can be detected in cell extracts for at least 21 d, suggesting that the exogenous gene is integrated. The low K+/Ca2+ transfection method is more effective than SrCl2 used as an alternative for CaCl2 in Ca2+ sensitive cells. Low K+ medium enhances cell survival for Ca2+ mediated transfection but also appears to have a beneficial effect on DNA uptake or expression.
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PMID:Expression of transfected DNA by primary murine keratinocytes. 245 57

We examine the effect of keratinocyte differentiation upon transient expression of a nonepithelial gene following DNA-mediated transfer. Cultures of primary epidermal keratinocytes were transfected with the reporter gene, chloramphenicol acetyltransferase (CAT). The CAT gene was linked at the 5' end to the long terminal repeat (LTR) regulatory sequences from Rous sarcoma virus, and gene transfer was accomplished by the calcium phosphate coprecipitation method. Transfected cells were fractionated on Ficoll 400 density gradients. The major finding of this study was that the larger, more differentiated cells displayed five- to seven-fold higher levels of CAT activity per cell than the smaller, less differentiated cells. The higher levels of CAT activity did not result from greater uptake of DNA because cells of all gradient fractions contained one to two copies of plasmid DNA per cell. Furthermore, the CAT gene linked to the regulatory sequences from another virus, SV40, gave the same result. We conclude that the CAT gene, when controlled by these viral regulatory sequences, is expressed more efficiently in differentiated keratinocytes. These results have important implications for the interpretation of future studies of gene expression in transfected keratinocytes.
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PMID:Transient expression of a transfected gene in cultured epidermal keratinocytes: implications for future studies. 246 54

Our previous work demonstrated that the inhibition of type I collagen synthesis by 1,25-dihydroxyvitamin D (1,25-(OH)2D3) in fetal rat calvaria and cultured rat osteosarcoma cells is accompanied by equivalent reduction in steady state levels of alpha 1(I) and alpha 2(I) collagen mRNA. To pursue the mechanism for this effect, we isolated and sequenced a 3.6-kilobase DNA fragment that contained the promoter for the rat alpha 1(I) collagen gene. This promoter fragment was fused to the chloramphenicol acetyltransferase gene and was introduced into ROS 17/2.8 cells by calcium phosphate co-precipitation. Expression of this construct was diminished by 1,25-(OH)2D3 to the same degree as the endogenous collagen gene in both transient expression assays and in permanently selected bone cells. However, a fibroblast cell line did not show a similar reduction in the activity of the transgene or the endogenous collagen gene. These experiments indicate that the alpha 1(I) promoter contains cis-active elements which are regulated by the 1,25-(OH)2D3 receptor in ROS 17/2.8 cells.
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PMID:Isolation and characterization of the rat alpha 1(I) collagen promoter. Regulation by 1,25-dihydroxyvitamin D. 820 63

We have constructed the expression plasmids harboring protein kinase C (PKC) mutant cDNAs with a series of deletions in the PKC coding region. These plasmids were transfected into COS7 cells to characterize the PKC mutants. Immunoblot analysis using the anti-PKC antibody identified proteins with the Mr values expected from the PKC mutant cDNAs in the extracts from COS7 cells. The wild-type PKC, when expressed in COS7 cells, conferred increased phorbol ester binding activity on intact cells; but the PKC mutants with the deletion around the C1 region did not show this activity. The wild-type PKC showed protein kinase activity dependent on phospholipid, Ca2+, and phorbol ester, whereas these PKC mutants exhibited protein kinase activity independent of the activators in a cell-free system. A PKC mutant cDNA with the deletion in the C2 region gave increased phorbol ester binding activity. Protein kinase activity of this mutant was much less dependent on Ca2+ compared with the wild-type PKC. A PKC mutant cDNA with the deletion in the C3 region conferred increased phorbol ester binding activity, but neither activator-dependent nor -independent protein kinase activity. These results indicate that elimination of the C1 region of PKC gives rise to constitutively active PKC independent of phospholipid, Ca2+, and phorbol ester and that the C1-C3 regions play distinct roles in the regulatory and catalytic function of PKC. In another series of experiments, transfection of some PKC mutant cDNAs with the deletions around the C1 region into Chinese hamster ovary and Jurkat cells activated the activator protein-1-binding element or the c-fos gene enhancer linked to the chloramphenicol acetyltransferase reporter gene in the absence of phorbol ester. Microinjection of these constructs into Xenopus oocytes induced initiation of germinal vesicle breakdown, indicating that they stimulated the PKC pathway in vivo. Thus, the phorbol ester-independent PKC mutant cDNAs could be a powerful tool to investigate the transmembrane signaling pathway mediated by PKC.
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PMID:Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C. 276 32

A simple method for introducing genes into respiratory epithelial cells would assist molecular studies of a variety of pulmonary disorders. Several different techniques for introducing foreign DNA into cells have been described but have either not been useful for respiratory epithelial cells or are difficult and cumbersome to perform. Electroporation is a simple technique that consists of exposing a cell-DNA suspension to an electric shock. Although it has been used to introduce genes into a variety of cell types, it has not previously been applied to respiratory epithelial cells. Human nasal epithelial cells were transfected with the plasmid pRSVCAT, which is an expression vector containing the origin of replication of pBR322 coupled to the Rous sarcoma virus (RSV) long terminal repeat (LTR) region driving the coding sequence for the chloramphenicol acetyltransferase (CAT) gene. The CAT gene is useful for determining optimal conditions for electroporation since it is not normally present in eukaryotic cells, and CAT activity correlates with the level of CAT mRNA; this provides a measure of expression of introduced foreign genes. Successful expression of the CAT gene was demonstrated by electroporation, whereas calcium phosphate transfection resulted in very low CAT activity. Optimal conditions for electroporation of respiratory epithelial cells were determined. Electroporating nasal epithelial cells using 500 volts, a DNA concentration of 10 micrograms/ml, and a sucrose buffer yielded the highest CAT activity, which peaked at 48 h after electroporation.
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PMID:The introduction of biologically active foreign genes into human respiratory epithelial cells using electroporation. 284 46

Insulin has been shown to inhibit rat growth hormone (GH) gene transcription. The effects of insulin were therefore tested on the expression of a transfected human GH gene. A 2.6-kilobase EcoRI fragment of the human GH gene was propagated in pUC18 and transfected by calcium-phosphate shock into HeLa and GC cells, respectively. Transfected cells grown in serum-free medium for 72 h expressed human GH measured by specific radioimmunoassay, incorporation of [35S] methionine into newly synthesized GH, and the presence of the appropriately sized protected transcripts seen after RNase protection assay. Immunoprecipitation analysis showed that insulin (0.7-7 nM) suppressed both the basal as well as the hydrocortisone (100 nM)-stimulated expression of newly synthesized 22-kDa GH in a dose-dependent fashion. Insulin (7 nM) also suppressed the basal and hydrocortisone-stimulated GH mRNA transcripts in these cells. Control nontransfected cells did not express human GH. Cells transfected with the truncated pOGH gene and pTKGH gene failed to respond to insulin treatment, whereas the human GH promoter was able to confer insulin responsiveness to the chloramphenicol acetyltransferase reporter gene. cis-Acting regulatory sequences residing on the 497-base pair 5'-flanking region of the human GH gene therefore appear to be a requirement for human GH gene response to the insulin signal.
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PMID:Insulin regulates expression of the human growth hormone gene in transfected cells. 290 52

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.
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PMID:Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression. 292 66


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