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)

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.
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PMID:Interferon-responsive regulatory elements in the promoter of the human 2',5'-oligo(A) synthetase gene. 283 Apr 97

We investigated the role of sequences flanking the transcription initiation site of the delta 1-crystallin gene in transient transfection assays of primary embryonic chicken lens epithelial cells or fibroblasts. Varying lengths of the 5' flanking sequence of the delta 1-crystallin gene (containing some untranslated sequence from exon 1) were fused to the bacterial chloramphenicol acetyltransferase (CAT) gene in the pSVOCAT plasmid. A plasmid carrying the bacterial beta-galactosidase gene driven by the Rous sarcoma virus (RSV) promoter was used as an internal control. Standardized results showed that the sequence located between -120 to -43 exhibited strong promoter activity; however, the promoter activity was markedly reduced (20-fold) when the upstream sequence between -603 and -120 was included in the construct. The delta 1-crystallin promoter displayed little lens preference. This upstream sequence did not reduce the activity of the Simian virus 40 (SV40) early promoter (with or without its enhancer) or the Herpes thymidine kinase promoter in transfection tests, indicating some specificity in its effect. Evidence for a delta 1-crystallin negative trans-acting factor was provided by competition experiments. Our data raise the possibility that expression of the delta 1-crystallin gene involves a negative cis-acting transcription element, a speculation which may deserve further attention in view of the gradual decrease in delta-crystallin synthesis in the developing lens.
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PMID:Evidence for positive and negative regulation in the promoter of the chicken delta 1-crystallin gene. 283 46

We used rep+ and rep- recombinant AAV-plasmid vectors containing the nonselectable marker chloramphenicol acetyltransferase (CAT) driven by the AAV p40 promoter, and having a selectable marker, neo, inserted in the plasmid genome, and driven by a herpesvirus thymidine kinase gene promoter. Each vector was transfected into human 293 cells or HeLa cells and the neo gene was used to select geneticin-resistant (genr) cells containing integrated vectors. The genr cells were then screened for expression of the unselected marker CAT. For 293 cells, most clones from the rep- vector gave high CAT expression whereas only 50% of those from the rep+ vector expressed CAT, generally at low level. For HeLa cells about 25% of the clones derived from either the rep+ or rep- vector expressed CAT, and several clones from the rep+ vector gave very high yields. We also analyzed integrated rep+ vectors by rescue after superinfection with adenovirus and by Southern blotting. The AAV-CAT genome could be rescued from 50% of HeLa cell clones but not from 293 cell clones. Lack of rescuability reflected rearrangement of the AAV genome termini or the rep gene. Western blotting showed low level constitutive expression of rep protein in one 293 cell clone and two HeLa cell clones. Thus, the AAV p40 promoter (as well as p5 and p19) can function in integrated vectors to express unselected markers which can subsequently be rescued. Expression and rescue depended upon several parameters including the cell type, the initial structure of the vector (rep+ or rep-) but not continued expression of rep, and possibly global effects of the surrounding chromatin.
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PMID:Expression and rescue of a nonselected marker from an integrated AAV vector. 284 41

The human basement membrane specific collagen type IV is a heterotrimer composed of two alpha 1(IV) chains and one alpha 2(IV) chain. A partial genomic EcoRI library was screened with cDNA clones representing the 5' end regions of the alpha 1(IV) and the alpha 2(IV) mRNA. A 2.2-kb genomic fragment was isolated and sequenced, which contains the 5' terminal exons of both genes located in close vicinity. The two genes were found to be arranged in opposite direction, head-to-head, separated only by a short region of 127 bp, apparently representing promoters of both genes as indicated by the existence of typical sequence motifs (CAT-box, SP1 consensus sequence). These data suggest that the alpha 1(IV) and alpha 2(IV) genes use a common, bidirectional promoter. The striking symmetrical arrangement of sequence elements within the promoter may be of basic importance for the coordination of bidirectional transcription. The promoter region had no detectable transcriptional activity in transient gene expression assays after fusion to the chloramphenicol acetylase (CAT) gene in either direction, indicating the necessity of additional elements for efficient and tissue-specific expression of both genes. Constructs containing different segments of both genes failed to identify regions with enhancing activity for the homologous collagen type IV promoter. When the heterologous HSV thymidine kinase promoter was used, a negatively acting region was identified. This indicates that the alpha 1(IV) and alpha 2(IV) promoter activity is controlled by additional regulatory elements present on distant portions of both genes.
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PMID:The genes for the alpha 1(IV) and alpha 2(IV) chains of human basement membrane collagen type IV are arranged head-to-head and separated by a bidirectional promoter of unique structure. 284 80

The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down
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PMID:The herpes simplex virus type 1 alpha protein ICP27 can act as a trans-repressor or a trans-activator in combination with ICP4 and ICP0. 284 67

High levels of expression of cloned genes have been obtained in mammalian cells by using poxvirus-derived insertion/expression vectors. These vectors employ the cis-acting element (CAE I) that directs the transcription of one of the most strongly expressed genes of cowpox virus. This gene (the 160K gene) encodes the 160-kDa protein that is the major component of the A-type cytoplasmic inclusions. Its counterpart in vaccinia virus (VV) is the 94K gene contained in the HindIII A fragment of the viral DNA. Two insertion vectors have been constructed; each is designed to allow cloned genes to be placed immediately downstream of a modified version of CAE I within a poxvirus genome. One vector, p1200, enables the CAE I-cloned-gene constructs to be inserted into the thymidine kinase gene of VV. This vector was used to create a VV recombinant that directed expression of the chloramphenicol acetyltransferase (CAT) gene. The other vector, p2101, enables the CAE I-cloned-gene constructs to be inserted into the VV 94K gene. The prototype of this vector was used to create a VV recombinant that directed expression of a hybrid CAT-lacZ gene. Infection of cultured human cells with these recombinants led to high levels of synthesis of either the CAT gene product or the CAT-lacZ gene product. Each of these proteins was produced in quantities that were easily detected by Coomassie blue staining of total cell proteins resolved by polyacrylamide gel electrophoresis. We estimate that these vectors are capable of directing the synthesis of milligram amounts of gene product per 10(9) mammalian cells.
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PMID:A poxvirus-derived vector that directs high levels of expression of cloned genes in mammalian cells. 284 5

We have isolated a c-erbA cDNA clone from a GH3 cell library. The clone, denoted erb62, is 4.5 kilobases long and encodes a 461-amino acid beta-type c-erbA protein. This c-erbA protein binds 3,5,3'-triiodothyronine (T3) and T3 analogs with affinities similar to those of the authentic T3 receptor. By RNA gel blot analysis, erb62 hybridizes to a 6-kilobase RNA found in organs that express T3 receptors--e.g., heart, kidney, and brain. A COS-cell transient cotransfection system was used to show that erb62 encodes a biologically active T3 receptor. An oligonucleotide, corresponding to a portion of the rat growth hormone gene 5'-flanking region that contains a T3 response element, was inserted on the 5' side of the herpes simplex virus thymidine kinase promoter in a chloramphenicol acetyltransferase-expressing plasmid. Reporter gene expression directed by this hybrid promoter was T3 inducible only if this plasmid was cotransfected with an erb62-expressing plasmid.
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PMID:Isolation of a cDNA clone encoding a biologically active thyroid hormone receptor. 289 22

We have extensively characterized the sequences of the rat growth hormone (rGH) promoter required for induction by T3 (thyroid hormone, 3,5,3'-L-triiodothyronine) in a transient transfection system. Oligonucleotides containing portions of the rGH promoter sequence with various deletions and point mutations were placed upstream of the first 137 base pairs of the rGH promoter or the heterologous herpes virus thymidine kinase promoter in chloramphenicol acetyltransferase expression vectors. The rGH137 and thymidine kinase promoters show no or minimal response to T3 in the basal state. The constructs were tested in GH4C1 rat pituitary cells and COS cells (functionally deficient in thyroid hormone receptor) with and without a co-transfected plasmid expressing a beta type c-erbA gene coding for a functional T3 receptor. Oligonucleotides containing the T3 receptor binding site confer hormone-dependent induction in a manner that is independent of either orientation or variation in position on the helix relative to the promoter. Point mutations in the sequence -189 to -173 result in loss of T3 induction, and bases between -173 and -167 were also required for a full T3 response. The minimal length to confer T3 induction to the rGH promoter was 23 base pairs (-190 to -167). Point mutations creating a perfect duplication of 7 base pairs within the receptor binding site conferred 12-fold T3 response to the rGH137 promoter, 3-fold greater than the wild type rGH237 construct. T3 inductibility was also transferred to the thymidine kinase promoter by an oligonucleotide containing the sequence -200 to -157, demonstrating that cell type specific elements located 3' to 157 of the rGH promoter are not required for thyroid hormone responsiveness.
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PMID:Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected beta type thyroid hormone receptor. 290 14

Regulation of gonadotropin gene expression by sex steroids may occur via direct effects on the pituitary and/or by indirect effects of steroid on the hypothalamus. To study direct estrogen regulation of the rat luteinizing hormone beta (LH beta) gene, we performed estrogen receptor-DNA binding studies and transient expression gene transfer experiments. Nitrocellulose filter binding studies were performed with purified estrogen receptor from calf uterus and labeled fragments of the LH beta gene. Dose-dependent specific binding to receptor occurred only with LH beta gene fragments containing a common 284-base region from -1388 to -1105 bases upstream from the transcriptional start site. This DNA region contained a 15-base imperfect palindromic region (GGACACCATCTGTCC) with sequence similarity to other estrogen-responsive elements. Biological function was tested by inserting portions of the 5'-flanking region of the gene next to the herpes simplex virus thymidine kinase promoter fused to the chloramphenicol acetyltransferase gene (LH beta-tkCAT) and performing gene transfer experiments with the pituitary GH3 cell line. Promoter activity in LH beta-tkCAT constructs containing LH beta gene sequences from bases -2013 to -613, or from bases -1388 to -613 in either orientation, exhibited stimulation with 17 beta-estradiol (E2) treatment; in contrast, constructs containing bases -885 to -613 were not regulated by E2. Positive regulation by E2 exhibited dose- and time-dependent stimulation, with a maximum 2- to 6-fold effect achieved after 48 h of treatment with 10(-8) M E2. The estrogen receptor appeared to be required for this biological response. Stimulation of LH beta-tkCAT constructs did not occur in L cells with undetectable levels of E2 receptor, but did occur after cotransfection of an LH beta-tkCAT construct and an expression vector containing the human estrogen receptor cDNA. These studies demonstrate that a 5'-flanking region of the rat LH beta gene can bind to the estrogen receptor and that this region can confer hormonal responsiveness to a heterologous promoter. Thus, positive steroid regulation of luteinizing hormone may occur directly on the pituitary at the level of the LH beta gene.
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PMID:An upstream region of the rat luteinizing hormone beta gene binds estrogen receptor and confers estrogen responsiveness. 290 46

We report a functional analysis of the human thymidine kinase (tk) gene promoter. We have linked the tk promoter to the chloramphenicol acetyltransferase (CAT) gene to allow direct measurement of promoter strength by assaying chloramphenicol acetyltransferase enzyme activity after transfection into mouse L cells. Putative transcription elements have been identified by deletion and mutation analysis of this promoter. The promoter relies primarily on two "CCAAT" elements and a series of "GC" elements found farther upstream. Two-thirds of promoter activity is maintained by a construct containing 139 base pairs of sequence upstream of the initiation of transcription that contains only one GC and one of the CCAAT elements. In addition, an evolutionary comparison identifies two highly conserved promoter elements: the -40 CCAAT element and a "TATA" element located at -21. We have further characterized both CCAAT elements using a mutational as well as protein binding analysis. From this study we have determined that both the -70 and -40 CCAAT elements bind strongly to the same factor, with a slightly higher affinity for the -40 CCAAT. Competition studies suggest that the CCAAT factor that binds to this promoter is homologous to protein nuclear factor Y, which binds to the major histocompatibility complex class II E alpha gene promoter. In addition, either CCAAT element is capable of supplying almost as much promoter strength as is supplied in the presence of both.
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PMID:The human thymidine kinase gene promoter. Deletion analysis and specific protein binding. 291 10


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