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)

Dopamine receptors are involved in many aspects of dopaminergic neurotransmission including regulation of motor control, cognition, affect and neuroendocrine function. The D1A receptor is the most widely distributed dopamine receptor in the brain and is expressed at high levels in the striatum and nucleus accumbens, but is also found throughout cortical, limbic, hypothalamic and thalamic brain regions. We have cloned a 6.4 kb fragment 5' of the human D1A dopamine receptor gene and shown that this region activates transcription of the chloramphenicol acetyltransferase (CAT) gene in a cell-specific manner. To study the expression of these sequences in vivo we analyzed the expression of the E. coli lac Z gene under the regulation of the 6.4 kb fragment in transgenic mice. Expression of the transgene was primarily detected in the brain, with only low levels detected in peripheral tissues. The 5' flanking sequences were able to direct the tissue-specific expression of lac Z in three different lines of transgenic mice, to a number of brain regions including the caudate-putamen, thalamus, amygdala, cerebral cortex, hippocampus and hypothalamus. Greatest expression of the lac Z gene was detected in areas of the thalamus and amygdaloid complex. In the striatum, beta-galactosidase activity was restricted to neurons within the matrix and was not detected within striosomes. Results of this study demonstrate that the 6.4 kb region upstream of the human D1A receptor gene is sufficient to confer tissue-specific expression in the CNS of transgenic mice. Furthermore, expression of the transgene to neurons within the matrix of the striatum, but not the striosomes suggests that expression of the D1A receptor may be regulated differently within these areas.
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PMID:The human D1A dopamine receptor gene promoter directs expression of a reporter gene to the central nervous system in transgenic mice. 763 83

We have established 15 separate lines of zebrafish transgenic for two plasmid DNAs containing Drosophila P element sequences and either the reporter genes chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), and beta-galactosidase (BGAL) or the reporter gene hygromycin phosphotransferase (HPT). Transient expression of CAT, but not NPT or BGAL, could be measured in the G0 (microinjected) generation, while expression in subsequent generations was below the limit of detection for CAT and NPT. All 15 lines contain stable low copy number integrations of between one and five transgene copies per cell with characteristic Southern blot "junction fragments" and have shown Mendelian inheritance after the G1 generation. The transgenes in 13 of 15 of the lines were originally inherited by about 10% or less of the G1 fish, suggesting a mosaic integration into the germline. Of 14 tested lines, none is associated with an embryonic recessive lethal phenotype as scored in the G3 generation.
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PMID:Inheritance of P element and reporter gene sequences in zebrafish. 770 14

The DNA sequences important for cell-specific expression and developmental regulation of corticotropin-releasing hormone (CRH) were analyzed in transgenic mice. A construct containing 0.5 kb of CRH 5' flanking DNA linked to the chloramphenicol acetyltransferase reporter gene was expressed in many brain regions and in several ectopic peripheral sites, suggesting that this portion of the CRH gene contains basal promoter activity but lacks DNA elements necessary for appropriate tissue specificity. Cell specificity of transgene expression was examined with a CRH-beta-galactosidase reporter construct containing the same 0.5-kb CRH promoter fragment, but also including the CRH structural gene and 2 kb of CRH 3' flanking DNA. Transgene expression was observed in inappropriate regions of the brain, but no expression was detected in peripheral tissues, suggesting that these additional CRH sequences suppress inappropriately high levels of peripheral expression. Cell-specific expression improved significantly with the inclusion of 8.7 kb of CRH 5' flanking DNA. Individual transgenic lines exhibited expression in a number of the major CRH neuronal groups including the paraventricular nucleus, medial geniculate nucleus, inferior olivary nucleus, and Barrington's nucleus. Transgene expression was properly activated in Barrington's nucleus during development. This study demonstrates that the regulatory control of cell-specific and developmentally appropriate CRH expression is complex, utilizing multiple DNA sequence elements located upstream and downstream of the CRH transcription start site.
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PMID:Expression of corticotropin-releasing hormone transgenes in neurons of adult and developing mice. 770 23

Loricrin gene expression is limited to terminally differentiating keratinocytes of stratified squamous epithelia. To define the regulatory elements that mediate the expression of the loricrin gene, we replaced the loricrin coding sequences from a 6.5-kilobase genomic fragment with the chloramphenicol acetyltransferase gene and transfected this construct into cultured mouse keratinocytes. High expression levels were observed in both undifferentiated as well as differentiating cells. Transgenic mice bearing a similar construct, but with beta-galactosidase as the reporter gene, corroborated these in vitro findings and showed tissue- and cell type-specific, but not differentiation-specific expression. Deletion analysis of the promoter region determined that sequences up to -60 base pairs from the start of transcription could be removed without significant loss of promoter activity. Within these proximal 60 base pairs is an evolutionarily conserved AP-1 element that is recognized by both purified c-Jun and AP-1 factors from keratinocytes in vitro. Mutation of this AP-1 site abolished the activity of the loricrin promoter. These studies show that elements directing expression of the loricrin gene to the stratified squamous epithelia are contained within a 6.5-kilobase genomic fragment, and those elements required to restrict expression to differentiated keratinocytes lie outside this region.
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PMID:The proximal promoter of the mouse loricrin gene contains a functional AP-1 element and directs keratinocyte-specific but not differentiation-specific expression. 773 16

An efficient method of transgene modulation in fertilized eggs has been developed that uses the Cre/loxP recombination system. Twelve transgenic mouse lines carrying a chicken beta-actin promoter-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase gene construct were produced. After selection of the line showing the highest expression of the CAT gene in a variety of tissues, eggs of this line were injected in the male or female pronucleus with a Cre expression vector placed under the control of the chicken beta-actin promoter and kept in a circular form to avoid genomic integration. This resulted in a transient expression of Cre in the eggs, leading to recombination of the transgene as detected by galactosidase expression and DNA analysis. Recombination was completed before the morula stage with both types of pronuclear injections and occurred with a very high frequency; no mosaicism, no incomplete recombination, and no integration of the Cre sequence were observed in 18 mice born with this modified transgene. The beta-galactosidase gene was expressed in various tissues at levels comparable to those found for the CAT gene in the founder line. This Cre transient expression system should be useful for breeding transgenic lines in which transgene expression leads to sterility or lethality--in particular, for selecting transgenic lines with high expression of a potentially lethal transgene whose full activity is difficult to explore in a conventional transgenic system because of the risk of selecting for transgenic lines carrying only poorly expressed transgenes.
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PMID:Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. 781 9

Vaccina virus (VV) and cowpox virus (CPV) differ in their abilities to replicate in Chinese hamster ovary (CHO) cells because VV has a disrupted host range (hr) gene. To facilitate an examination of the molecular events associated with abortive infection of CHO cells with VV, we constructed two sets of recombinant viruses that contain a viral early promoter regulating the cat gene encoding chloramphenicol acetyltransferase and viral intermediate or late promoters regulating the lacZ gene encoding beta-galactosidase. The first set has the disrupted hr gene and the second set has the intact CPV homolog, allowing replication in CHO cells. Reporter chloramphenicol acetyltransferase and beta-galactosidase assays demonstrated that early gene expression was unperturbed, whereas intermediate and late gene expression were severely inhibited under abortive conditions. Metabolic labeling studies confirmed the absence of viral late protein synthesis. The accumulation of viral DNA under abortive conditions was consistent with the synthesis of viral early proteins and established that inhibition of late protein synthesis was not primarily due to a replicative block. Analysis of steady state levels of viral mRNAs revealed substantial quantities of early and intermediate species but only very small amounts of late mRNAs under nonpermissive conditions. Despite the presence of viral intermediate mRNAs, the corresponding intermediate proteins, which function as late transcription factors, were not detected by immunoprecipitation of lysates from metabolically labeled infected CHO cells. Furthermore, when expression of lacZ was regulated by an intermediate promoter, no beta-galactosidase was detected even though lacZ transcripts were present. Thus, the abortive phenotype in CHO cells can be explained by a block to translation of intermediate mRNAs which prevents the synthesis of late transcription factors.
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PMID:Restriction of vaccinia virus replication in CHO cells occurs at the stage of viral intermediate protein synthesis. 785 9

Tyrosine hydroxylase (TH), the first and rate-limiting enzyme in the biosynthesis of catecholamine neurotransmitters, is expressed within central and peripheral catecholaminergic cells. To delineate DNA sequences necessary for tissue-specific expression of the rat TH gene, transgenic mice were produced containing 0.15 kb, 2.4 kb, and 9.0 kb of 5' flanking sequence fused to the E. coli lacZ (beta-galactosidase) reporter gene. The reporter gene expression in the transgenic animals was monitored by both X-gal histochemical staining and beta-galactosidase immunohistochemistry and compared to TH mRNA and protein expression. Transgenic mice bearing 9.0 kb, but not the smaller constructs with either 2.4 kb or 0.15 kb of 5' flanking sequence, fused to lacZ were able to direct high level expression of beta-galactosidase at levels equivalent to the endogenous TH in central catecholaminergic cells, and to a lesser degree to adrenal gland. Previously, 4.8 kb of 5' flanking region was reported to contain some tissue-specific element(s) determined by chloramphenicol acetyltransferase (CAT) assay using regional brain dissections and was not able to demonstrate cellular localization of the CAT expression [2]. Using histological procedures which allow for spatial resolution, this study demonstrated that the crucial catecholaminergic neuron-specific DNA element(s) resides between -9 kb and -2.4 kb of the 5' flanking region of the rat TH gene; this assertion is substantiated by the high-level of tissue-specific expression of lacZ in catecholaminergic cells.
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PMID:5' upstream DNA sequence of the rat tyrosine hydroxylase gene directs high-level and tissue-specific expression to catecholaminergic neurons in the central nervous system of transgenic mice. 789 12

Transgenic Xenopus laevis embryos were produced by transplantation of transfected cultured cell nuclei into unfertilized eggs. A Xenopus cell line, X-C, was stably transfected with plasmids containing a hygromycin-resistance gene and genes for either beta-galactosidase with a heat shock promoter or chloramphenicol acetyltransferase (CAT) with a muscle-specific actin promoter. Nuclei transplanted from these cells into unfertilized eggs directed development of embryos containing stably integrated copies of the plasmids in each cell. Transgenic embryos showed somite-specific expression of CAT and uniform expression of beta-galactosidase. Transgenic embryos produced by nuclear transplantation should be useful for testing the function of cloned genes in amphibian development.
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PMID:Transgenic X. laevis embryos from eggs transplanted with nuclei of transfected cultured cells. 793 20

Direct injection of DNA expression vectors into muscle leads to expression of encoded recombinant gene products in mature muscle cells. This phenomenon is not shared by most other organs. We have surveyed various organs in the rabbit to identify other cell types that would express DNA vectors after direct injection. We observed that thyroid follicular cells were capable of acquiring plasmid DNA and expressing recombinant gene products after direct interstitial injection of plasmid vectors into the thyroid gland. The level of expression of a chloramphenicol acetyltransferase (CAT) reporter gene in thyroid tissue was similar to that seen in muscle tissue three days after injection in controlled experiments. Using a beta-galactosidase reporter gene, expression was localized to thyroid follicular cells. CAT activity decreased with first-order kinetics and a half-life t1/2 of 40 hr. DNA was identified in thyroid tissue by polymerase chain reaction (PCR) analysis and displayed first-order elimination kinetics with a half-life t1/2 of 10 hr. The persistence of the gene and gene product in the thyroid was significantly different from that observed after injection of DNA vectors into muscle or delivery of DNA vectors to the liver using asialoglycoprotein/polylysine/DNA complexes, suggesting that there are significant differences in the process of DNA uptake or compartmentalization in these experimental systems. These results introduce the possibility of developing the thyroid as a novel target for treating certain thyroid or systemic diseases using DNA vectors.
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PMID:In vivo gene transfer into rabbit thyroid follicular cells by direct DNA injection. 798 8

We developed a convenient method to methylate all CpG dinucleotides in both strands in a selected region of a plasmid, and investigated the effect of DNA methylation in the transcribed regions of reporter genes on the transient expression in HeLa cells. In a construct containing the chloramphenicol acetyltransferase (CAT) gene linked to the SV40 early promoter, methylation in the CAT structural gene repressed CAT activity. Methylation in the transcribed region of the Escherichia coli lacZ gene driven by the human cytomegalovirus immediate early promoter also inhibited expression of beta-galactosidase activity. These results suggest that methylation in the transcribed region as well as promoter methylation may affect transcription.
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PMID:Repression of transient expression by DNA methylation in transcribed regions of reporter genes introduced into cultured human cells. 799 98


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