Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As has been demonstrated for herpes simplex virus type 2, we show in this report that the herpes simplex virus type 1 ribonucleotide reductase large subunit (RR1) gene is trans activated in transient transfection assays by VP16 and ICP0 but not by ICP4. Deletion analysis demonstrated that responsiveness to induction to VP16 resides in an octamer/TAATGARAT sequence of the RR1 promoter and that the TATA box alone is sufficient to provide induction by ICP0. The induction of the RR1 gene by ICP0 but not by ICP4 suggested that it might be possible to identify the cis-acting element(s) responsive to ICP4 in an ICP4-inducible promoter. To this end, a series of chimeric promoters containing various portions of the regulatory sequences of the RR1 promoter and thymidine kinase (TK) promoter were constructed. The TK promoter is trans activated by both ICP0 and ICP4 in transient transfection assays and by ICP4 in infection. The data show that replacing the RR1 TATA region with the TK TATA region permits ICP4 inducibility even if the rest of the RR1 promoter elements remain intact. To test whether the RR1 gene is induced by ICP0 during infection, four mutant viruses were constructed. (i) TAATGARAT+ has the wild-type RR1 promoter driving chloramphenicol acetyltransferase (CAT) and the RR2 promoter driving the lacZ gene. The RR2 gene codes for the small subunit of the ribonucleotide reductase and is expressed as a beta gene. (ii) TAATGARAT- has a triple-base change in the octamer/TAATGARAT element which renders it unresponsive to VP16 trans activation, eliminating that portion of the activation of the RR1 gene. (iii) TAATGARAT- delta alpha 0 has a deletion of the alpha 0 gene. (iv) TAATGARAT- delta alpha 4 has a deletion of the alpha 4 gene. Infections were carried out in Vero cells at a multiplicity of infection of 10 per cell; cells were assayed for CAT and beta-galactosidase (beta-Gal) activities and for virus yields. The first two infections gave strong CAT and beta-Gal activities and high yields of progeny virus. Infection with the third virus showed no CAT activity but did produce high levels of beta-Gal activity and virus progeny. The fourth infection resulted in strong CAT activity but no beta-Gal activity or progeny virus. The data demonstrated that the RR1 promoter was activated in the absence of ICP4 but not in the absence of ICP0 in these infections.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:The RR1 gene of herpes simplex virus type 1 is uniquely trans activated by ICP0 during infection. 839 74

The lasR gene of Pseudomonas aeruginosa is required for transcription of the genes for elastase (lasB) and LasA protease (lasA), two proteases associated with virulence. We report here that the alkaline protease gene (apr) also requires the lasR gene for transcription. Alkaline protease mRNA was absent in the lasR mutant PAO-R1 and present when an intact lasR gene was supplied in trans as determined by Northern (RNA) analysis. The lasR gene also enhances exotoxin A production. Exotoxin A activity in supernatants of PAO-R1 were 30% less than in supernatants of the parental strain, PAO-SR. Multiple copies of lasR in trans in PAO-R1 in increased toxin A activity to twice the parental levels. Analysis of PAO-R1 containing the toxA promoter fused to beta-galactosidase suggests that LasR acts at the toxA promoter or at upstream toxA mRNA sequences. beta-Galactosidase activity was approximately 40% lower in PAO-R1 than in the parental strain, PAO-SR. Furthermore, the effect of LasR on the toxA promoter is not due to the stimulation of transcription of regA, a transcriptional activator of toxA. No difference in chloramphenicol acetyltransferase (CAT) activity was noted between PAO-SR and PAO-R1 containing transcriptional regA promoter-CAT gene fusions. These results broaden the regulatory dominion of lasR and suggest that the lasR gene plays a global role in P. aeruginosa pathogenesis.
 ...
PMID:LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. 845 22

A new Escherichia coli expression vector with increased stability was developed based on bacteriophage Mu. Unlike traditional expression vectors, the vector described herein is chromosome based rather than existing as an autonomously replicating plasmid. The chromosomal location resulted in extreme stability of the vector even in the absence of selective pressure. Both replication and heterologous protein synthesis could be induced by temperature shift. Expression of the heterologous gene was controlled by the Mu middle promoter and was dependent on the presence of the transactivator, Mor, of the Mu middle promoter. Four proteins, beta-galactosidase, chloramphenicol acetyltransferase, porcine somatotropin and human growth hormone, were made from this vector at levels ranging from 5 to 20% of total cell protein. Expression from the middle promoter was highest when inductions were done in rich media. The expression of some genes varied in different strains.
 ...
PMID:A chromosomal expression vector for Escherichia coli based on the bacteriophage Mu. 847 59

The stimulation of reporter gene expression following co-transfection with the S4 gene of mammalian reoviruses was analysed. The sigma 3 protein of type 3 reovirus gave a five- to eightfold increase in expression of chloramphenicol acetyltransferase and beta-galactosidase but this was found to be dependent upon the nature of the promoter being used to drive reporter gene expression. The sigma 3 protein of reovirus type 1 failed to stimulate reporter gene expression under any of the conditions used. Hybrid constructs between the S4 genes of reoviruses type 1 and 3 were used to map the stimulation characteristic to the carboxy-terminal third of the gene. Analysis of the level of sigma 3 protein accumulation in transfected cells showed that the reovirus type 3 protein accumulated to a much higher level than that of reovirus type 1. Using the hybrid gene constructs this higher level of protein accumulation was shown to co-segregate with the ability to stimulate reporter gene expression.
 ...
PMID:Analysis of the stimulation of reporter gene expression by the sigma 3 protein of reovirus in co-transfected cells. 850 59

The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the third instar to the end of adult life. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. To study the molecular basis for ecdysone regulated Lsp-2 activity, deletion mutants of the Lsp-2 5'-flanking region were constructed by fusion to either the Escherichia coli chloramphenicol acetyltransferase (CAT) gene or to an hsp70-lacZ hybrid gene encoding beta-galactosidase. Constructs transfected into Drosophila S2/M3 cells were shown to confer transient ecdysone inducibility on the reporter genes. A single functional ecdysone response element (EcRE) was localized at position -75 relative to the Lsp-2 transcription initiation site. In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27-bp sequence harboring the EcRE bound both the Drosophila ecdysone receptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a cooperative manner. Competition experiments indicate that the affinity of the Lsp-2 EcRE for the ecdysone receptor complex is comparable to that of the canonical EcRE of the hsp27 gene and is at least 4-fold greater than that of Fbp1, another fat body-specific Drosophila gene. Our results suggest that structural features of this EcRE determine its ability to induce ecdysone responsiveness at a lower ligand concentration and may form the basis for differential hormone responsiveness within the fat body.
 ...
PMID:Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene. 854 20

The transgenic mouse system is a powerful tool for the study of gene function. However, when the analysis involves genes that are critical for the normal developmental process, the usefulness of transgenic mouse systems is limited (for review see Hanahan, 1989; Westphal and Gruss, 1989; Byrne et al., 1991). This is due to potential transgene interference with development in case of ectopic or high level expression. As a result, establishing permanent transgenic mouse lines expressing these types of genes has proven difficult. To circumvent these difficulties, a binary transgenic mouse system has been established, termed the Multiplex System (Byrne and Ruddle, 1989). This is a two-tiered gene activation system in which expression of the gene of interest occurs only in offspring carrying transgenes encoding both components: transactivator and transresponder. Transactivator lines contain the gene encoding the VP16 protein of herpes simplex virus. Transresponder lines harbour the gene of interest linked to the IE promoter which includes recognition sequences for the VP16 transactivator. Previously, the inducibility of a chloramphenicol acetyltransferase reporter gene in newborn offspring that carried both a transactivator and transresponder transgene (Byrne and Ruddle, 1989) has been shown. Moreover, it has been demonstrated that expression of the VP16 protein was not detrimental to development and that transactivation appeared to be tissue specific. Here, the potential of the system for the expression of transgenes in early mouse embryogenesis was examined, using the Escherichia coli beta-galactosidase gene as a reporter in the transresponder mouse strain. To direct expression of VP16, the murine Hoxc-8 promoter, which is known to be active during early development, was used. Embryos from crosses of transactivators to transresponders were isolated at different stages of development and stained for beta-galactosidase activity. Transactivation, as demonstrated by strong beta-galactosidase staining, could be detected as early as eight days of development. At all stages examined, the pattern of lacZ transresponder gene expression accurately reflected the activity of the Hoxc-8 promoter controlling VP16 expression. It is demonstrated that the Multiplex System can be used to express transresponder transgenes in a spatially and temporally defined manner in multiple cell types early during mouse embryogenesis.
 ...
PMID:Spatial and temporal regulation of a lacZ reporter transgene in a binary transgenic mouse system. 858 38

In order to develop systems to express mammalian proteins in human skin-derived cells, we tested 6 different viral and 1 eukaryotic promoter (pCMV, pRSV, pSV, pMMTV, pPoly E, pPoly L, pHMT) for their ability to drive the expression of the chloramphenicol acetyltransferase (CAT) enzyme in different human skin-derived cells. DNA was transfected in human keratinocytes derived from normal foreskin and cervix, in the HPV-negative cervical cancer line HT-3 and in malignant melanoma cell lines (SK-Mel 23, SK-Mel 37) using a liposome-based technique or calcium precipitation. Transfection efficacy was controlled by cotransfection of a beta-galactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined by incubation of the cell extract prepared from the transfected cells with 14 C-labeled chloramphenicol. The CMV-promoter was highly active in all skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, the RSV-, SV-, and HMT-promoter were less active and varied in dependence of the cell type. The pattern of the promoter activity differed between benign and transformed genital keratinocytes. Only the SV-promoter showed a comparable strong basal activity, which was restricted to the SK-Mel 37 cells. In conclusion, the promoter activity has to be tested for each cell type depending on the aims of the gene expression.
 ...
PMID:Differential promoter activity in benign and malignant human cells of skin origin. 858 24

The expression of myogenin is suppressed during innervation and has been implicated in determining properties of skeletal muscle which are regulated by electrical activity. We previously reported that transcription driven by 3700 bp of the mouse myogenin upstream sequence (MYG3700) is activated by denervation in transgenic mice (Nucleic Acids Res. 21, 5684-5693, 1993). To extend our investigation of the activity dependence of the myogenin promoter, we have utilized myoblast implantation as a novel approach to in vivo reporter analysis. Myoblasts for hindlimb injections were generated by stable transfection of chloramphenicol acetyltransferase (CAT) reporters into a beta-galactosidase-expressing line of C2 cells. In vitro characterization of stable myoblast clones carrying myogenin-CAT deletion constructs revealed that while the proximal myogenin 5'-flanking sequence confers myotube specificity, high-level expression requires a region upstream (-335 to -1102) which depends on chromosomal integration for its function. For analysis by implantation, incorporation of injected myoblasts into existing myofibers was confirmed by histochemical staining. Using clonal myoblasts harboring nicotinic receptor alpha-subunit (alpha 800) and myosin light chain receptors as positive and negative controls, respectively, for denervation responsiveness, we determined that the nuclei of injected myoblasts are susceptible to regulatory signals imposed by nerve-induced electrical activity of the myofiber into which they incorporate. In in vivo analysis of myogenin upstream sequence by implantation, CAT activities of MYG3700 and MYG1565 reporters in injected limbs increased up to fourfold within 4 days after denervation, whereas the activities of MYG1102 and MYG335 were unchanged. By 10 days after denervation, all myogenin reporters displayed denervation responsiveness. These implantation data suggest an early phase of denervation activation, one that is mediated by control elements residing within -1102 to -1565 of the myogenin upstream sequence. Thus, the combined analyses of stable reporter myoblast lines in vitro and in vivo by implantation provide an efficient means of evaluating regulatory regions for high-level expression and neural modulation of muscle gene transcription.
 ...
PMID:Analysis of neural-responsive myogenin upstream sequences by myoblast implantation. 861 76

The bet regulon allows Escherichia coli to synthesize the osmoprotectant glycine betaine from choline. It comprises a regulatory gene, betI, and three structural genes: betT (choline porter), betA (choline dehydrogenase), and betB (betaine aldehyde dehydrogenase). The bet genes are regulated by oxygen, choline, and osmotic stress. Primer extension analysis identified two partially overlapping promoters which were responsible for the divergent expression of the betT and betIBA transcripts. The transcripts were initiated 61 bp apart. Regulation of the promoters was investigated by using cat (chloramphenicol acetyltransferase) and lacZ (beta-galactosidase) operon fusions. Mutation of betI on plasmid F'2 revealed that BetI is a repressor which regulates both promoters simultaneously in response to the inducer choline. Both promoters remained inducible by osmotic stress in a betI mutant background. On the basis of experiments with hns and hns rpoS mutants, we conclude that osmoregulation of the bet promoters was hns independent. The bet promoters were repressed by ArcA under anaerobic growth conditions. An 89-bp promoter fragment, as well as all larger fragments tested, which included both transcriptional start points, displayed osmotic induction and BetI-dependent choline regulation when linked with a cat reporter gene on plasmid pKK232-8. Flanking DNA, presumably on the betT side of the promoter region, appeared to be needed for ArcA-dependent regulation of both promoters.
 ...
PMID:The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress. 862 94

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.
 ...
PMID:GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. 864 9


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>