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 pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the -10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the -10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gel-mobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.
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PMID:Regulation of the phosphate regulon of Escherichia coli: characterization of the promoter of the pstS gene. 265 88

The phoB gene product of Escherichia coli is the transcriptional activator for the genes in the phosphate regulon as well as for phoB itself, all of which are induced by phosphate starvation. The phoR gene product modulates PhoB function in response to the phosphate concentrations in the medium. We quantitatively compared the levels of expression of the phoA, phoB, phoE, and pstS genes in several phoB mutants with different phenotypes by constructing operon fusions of these genes with the gene for chloramphenicol acetyltransferase. Although all the phoB mutants examined had little activator function for phoA, three among the four mutants showed various levels of the activator function for phoB, pstS, and phoE. To study the functional motifs of the PhoB and PhoR proteins, we cloned and sequenced the four classical phoB and six phoR mutant genes. All of the phoB mutations and one of the phoR mutations were missense mutations, and most of the altered amino acids were in the highly conserved amino acids among the regulatory proteins homologous to PhoB or PhoR protein, such as the OmpR, SfrA, and VirG proteins or the EnvZ, CpxA, and VirA proteins. The other five phoR mutations were nonsense mutations.
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PMID:Regulation of the phosphate regulon of Escherichia coli: analysis of mutant phoB and phoR genes causing different phenotypes. 267 81

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

The ability of recombinant DNA viruses to transfer genes into hematopoietic cells has been explored. A recombinant simian virus 40 (SV40) in which the early region had been replaced with the chloramphenicol acetyltransferase (CAT) gene driven by the promoter from Rous sarcoma virus (RSV), was constructed. This virus transferred the CAT gene more efficiently into mouse and human bone marrow cells and into the K562, MEL, and WEHI hematopoietic tissue culture cell lines, than the classical calcium phosphate DNA transfer procedure, as shown by assay for CAT activity 48 hr after infection. Recombinant SV40 virions were also shown to be capable of stably transforming Chinese hamster ovary cells by use of an early region recombinant containing the methotrexate-resistant dihydrofolate reductase (DHFR) gene driven by the RSV promoter. The entire DHFR transcriptional unit could be detected in the genome of transformed cells that were also shown to be resistant to methotrexate. A recombinant adenovirus stock containing the neomycin-resistance gene driven by the SV40 early promoter was used to infect the K562 and MEL hematopoietic cell lines to resistance to the antibiotic G418. Transformation frequency was 10- to 100-fold higher than that obtained with calcium phosphate-precipitated DNA. Most or all of the recombinant adenovirus genome was integrated as 1-3 copies in the transformed cells. These studies show the feasibility of using DNA viruses for introduction of new genetic material into hematopoietic cells.
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PMID:Transfer of genes into hematopoietic cells using recombinant DNA viruses. 298 41

Ornithine transcarbamylase (OTCase) is a mitochondrial matrix enzyme that catalyzes the 2nd step in the mammalian urea cycle. The gene encoding OTCase is located on the X chromosome and expression of OTCase is limited almost exclusively to hepatocytes. We have characterized a lambda phage recombinant, isolated from a mouse genomic library, that spans the first two exons of the mouse OTCase gene. Nuclease S1 mapping and primer extension analysis of this clone allowed us to determine that the transcription start site is 136 base pairs (bp) upstream from the translation initiation codon. Two TATA-like sequences were found 25 and 153 bp from the transcription initiation point. An 800-bp fragment containing the 5' flanking region of the OTCase gene was fused upstream to the coding sequence of the chloramphenicol acetyltransferase gene to assay promoter activity. This plasmid was introduced into mouse fibroblast NIH 3T3 cells and human hepatoma Hep G2 cells by the calcium phosphate co-precipitation method. After DNA transfection chloramphenicol acetyltransferase activity was observed only in Hep G2 cells. We conclude that this 800-bp fragment contains sufficient information to control OTCase gene expression in a tissue-specific manner, probably by interacting with trans-acting factor(s) which are not present in the other cell line.
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PMID:The 5' flanking region of the ornithine transcarbamylase gene contains DNA sequences regulating tissue-specific expression. 301 88

Recent studies have demonstrated that the left-handed, Z-DNA conformation is favored in polymers containing alternating purine/pyrimidine sequences that can exist in vivo and may play a role in gene expression. On the basis of this assumption, we have studied the effect of various cotransfected polynucleotides on the transient expression of the chloramphenicol acetyltransferase (CAT) gene in thymidine kinase-deficient murine L cells. Cotransfections were performed by calcium phosphate coprecipitation of CAT gene plasmids with various polymers, and the CAT enzymatic activity was measured in cell lysates after 48 hr. About 2- to 10-fold stimulation of CAT gene expression was observed when the cells were cotransfected with 10 micrograms (per 10-cm culture dish) of plasmid pSV2cat, which contains simian virus 40 (SV40) promoter and enhancer sequences, and 2-10 micrograms of polymers that can form Z-DNA, such as poly(dG-m5dC) X poly(dG-m5dC) or poly(dG-dC) X poly(dG-dC), as compared to transfection with pSV2cat alone. Further, enhanced CAT gene expression was also observed when cotransfections were performed with these polymers and two other plasmid vectors, one containing the SV40 promoter but no enhancer and the other lacking any SV40 regulatory sequences. However, poly(dA-dC) X poly(dG-dT), which can form Z-DNA, did not induce any stimulation. Similarly, no or very little stimulation was observed after cotransfection of pSV2cat with either poly(dG) X poly(dC) or poly(dA-dT) X poly(dA-dT), which do not adopt the Z conformation. These results suggest that certain polynucleotides may enhance transcription of the CAT gene.
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PMID:Enhanced expression of the bacterial chloramphenicol acetyltransferase gene in mouse cells cotransfected with synthetic polynucleotides able to form Z-DNA. 301 24

Introduction of DNA into human hemopoietic cells is required for the study of regulatory mechanisms operating in these cells, as well as for possible procedures of gene therapy. However, with hemopoietic cells the conventional technique of calcium phosphate precipitation is inefficient. The pathway of encapsidation of plasmid DNA as simian virus 40 (SV40) pseudovirions for the introduction of new genetic material was therefore investigated. Encapsidation was achieved in COS (monkey kidney) cells, which express SV40 large tumor (T) antigen constitutively. The vector, pSO, was introduced to the COS cells by DNA transfection. It carried the SV40 origin of replication (ori), to facilitate replication of the plasmid in the COS cells. The SV40 capsid proteins were supplied in trans by a helper SV40 virus. The bacterial chloramphenicol acetyltransferase gene cat was used as a model for gene transmission. After encapsidation, the pseudovirions were used in infection of the human erythroleukemic cell line K562 and of normal human bone marrow cells. The results demonstrate that the cat gene can be transmitted with high efficiency. Over 40% of the infected K562 cells and 30% of the infected bone marrow cells were observed to contain plasmid DNA 48 hr after infection. Moreover, the results suggest that the efficiency of gene transmission by this vector can be improved and so may approach the theoretical 100%.
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PMID:Efficient introduction of plasmid DNA into human hemopoietic cells by encapsidation in simian virus 40 pseudovirions. 301 51

Expression of the human renin gene is regulated in a tissue-specific manner, but study of this regulation has been limited by a lack of suitable cell lines that simulate endogenous control. In order to characterize the regulation of renin gene expression, the 5' flanking region (892 base pairs) from the human renin gene was linked to the chloramphenicol acetyltransferase gene and was introduced into multiple human cell lines by calcium phosphate precipitation or electroporation methods to assess transcriptional control. The human renin promoter was active when transfected into cultured human choriocarcinoma cells (JEG-3) and rat vascular smooth muscle cells, but it was not active in many other cloned cell types. These results suggest that selective cell lines contain the specific trans-acting factors necessary for human renin gene expression, and support the concept of cell-specific expression of this gene.
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PMID:Functional human renin promoter in transfected cells: evidence for cell-specific expression. 307 83


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