Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The phage DNA-directed synthesis of beta-galactosidase has been examined in a system containing the following purified Escherichia coli factors: RNA polymerase; cyclic AMP receptor protein; N10-formyltetrahydrofolate Met-tRNAf transformylase; initiation factors 1, 2, and 3; elongation factors Tu, Ts, and G; release factors 1 and 2; 20 aminoacyl-tRNA synthetases; L factor (Kung, H. F., Spears, C., and Weissbach, H. (1974) J. Biol. Chem. 250, 1556-1562); and Lalpha (Kung, H.-F., Spears, C., and Weissbach, H. (1976) Fed. proc. 35, 1537). Under these conditions, beta-galactosidase synthesis occurs at less than 1% of the rate obtained with unfractionated extracts, which suggested that other required components were lacking. The difficulty in obtaining large amounts of the purified aminoacyl-tRNA synthetases for these studies made it necessary to modify the system. It was possible to conserve many of the purified aminoacyl-tRNA synthetases since at least 13 of them could be replaced by an Ehrlich ascites extract. The ascites extract plus other E. coli purified factors was used as a basic system to search for additional components required for beta-galactosidase synthesis. The present report describes the purification from E. coli extracts of three fractions, called Lbeta, Lgamma, and Ldelta, that are needed to restore enzyme synthesis.
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PMID:DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors. 56 Oct 72

Mutations in the ARD1 gene prevent yeast cells from displaying G1-specific growth arrest in response to nitrogen deprivation and cause MATa haploids (but not MAT alpha haploids) to be mating defective. Analysis of cell type-specific gene expression by examination of RNA transcripts and measurement of beta-galactosidase activity from yeast gene-lacZ fusions demonstrated that the mating defect of MATa ard1 mutants was due to an inability to express genes required by MATa cells for the mating process. The lack of mating-specific gene expression in MATa cells was found to be due solely to derepression of the normally silent alpha information at the HML locus. The cryptic a information at the HMR locus was only very slightly derepressed in ard1 mutants, to a level insufficient to affect the mating efficiency of MAT alpha cells. The preferential elevation of expression from HML over HMR was also observed in ard1 mutants which contained the alternate arrangement of a information at HML and alpha information at HMR. Hence, the effect of the ard1 mutation was position specific (rather than information specific). Although the phenotype of ard1 mutants resembled that of cells with mutations in the SIR1 gene, both genetic and biochemical findings indicated that ARD1 control of HML expression was independent of the regulation imposed by SIR1 and the other SIR genes. These results suggest that the ARD1 gene encodes a protein product that acts, directly or indirectly, at the HML locus to repress its expression and, by analogy, may control expression of other genes involved in monitoring nutritional conditions.
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PMID:The yeast ARD1 gene product is required for repression of cryptic mating-type information at the HML locus. 331 86

To identify genes important in fat-cell metabolism and development, we have screened Drosophila stocks carrying an engineered transposable element that can reveal the presence of nearby enhancer elements. We have identified those "enhancer-trap lines" that contain transposable P elements integrated near fat-cell specific enhancer elements. We anticipate that the genes associated with these enhancers will provide information concerning fat-cell function and serve as target genes for studying fat-cell specific gene expression. Furthermore, the identification of enhancer-trap lines active in the developing fat cell should provide an entry point into the molecular and genetic analysis of early fat-cell development. Analysis of two lines has revealed that the transcription factors svp, a steroid-hormone receptor, and Kr, a zinc-finger protein, are present in the fat body; these factors are likely to be involved in fat-cell gene expression. In two other lines, beta-galactosidase was detected in a subset of adepithelial cells that may be the precursors to the adult fat cell. And finally, in a single line transgene activity is present in the progenitor cells of the embryonic fat body. The genes associated with these enhancer-trap lines may be involved in fat-cell development.
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PMID:Identification of fat-cell enhancer activity in Drosophila melanogaster using P-element enhancer traps. 755 62

Plastins are a family of human actin-binding proteins (isoforms) which are abundantly expressed in all normal replicating mammalian cells. One isoform, L-plastin, is constitutively expressed at high levels in hemopoietic cell types while T-plastin is constitutively expressed in all non-hemopoietic cells of solid tissues that have replicative potential (fibroblasts, endothelial cells, epithelial cells, melanocytes, etc.). L-plastin is, however, constitutively synthesized in many types of malignant human cells of solid tissues suggesting that its expression is induced during tumorigenesis. The frequency of L-plastin induction in some cancers of the steroid-regulated female reproductive tract (breast, ovary, uterus, and placenta) appears to be especially high (79% in a limited survey). To learn the mechanism of L-plastin gene activation accompanying tumorigenesis, we have begun to characterize the promoter and regulatory elements of the L-plastin gene. Transcription initiation from this promoter was found to occur at multiple sites and as near as 10 base pairs from the 3'-side of the TATAAA box. The promoter and its flanking DNA were cloned and sequenced to identify potential regulatory elements that participate in the induction of the L-plastin gene in neoplastic cells. Examination of upstream sequences revealed the existence of two potential progesterone, one potential estrogen, and four potential Ets-1 responsive elements flanking the promoter. A 315-base pair fragment spanning the TATAAA box and a potential Sp1-binding site exhibited maximum promoter activity using CAT as a reporter while longer promoter fragments extending into upstream flanking sequences spanning the hormone receptor-response elements exhibited reduced promoter activity. An expression vector, pHLPPr-1-neo, was constructed using a 5.1-kilobase pair EcoRI-HindIII fragment of the L-plastin gene that contained the potential upstream regulatory elements, the TATAAA box, and part of the first exon. This promoter could direct the constitutive expression of the reporter beta-galactosidase at high frequency in transfected colonies of transformed cells that express L-plastin constitutively; by contrast, this promoter was virtually inactive in transfected colonies of normal fibroblasts and it exhibited a low frequency of constitutive activation in transfected colonies of in vitro SV40-transformed fibroblasts which did not exhibit L-plastin expression. The utility of this recombinant promoter in determining the mechanism(s) that leads to activation of the L-plastin gene in tumor cells is discussed. The potential significance of regulation of the L-plastin gene by reproductive hormones in cancers arising in hormone-responsive tissues is also discussed.
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PMID:Characterization of the human L-plastin gene promoter in normal and neoplastic cells. 842 53

HMR 3004 is a new hydrazono ketolide characterized by a 3-keto function instead of the cladinose moiety. The effect of this antimicrobial agent on inducible and constitutive macrolide-lincosamide-streptogramin B (MLSB) resistance was tested in a lacZ reporter system under control of several ermAM-like attenuator variants. For one constitutively resistant Streptococcus agalactiae strain, three inducibly resistant Streptococcus pneumoniae strains, and one inducibly resistant Enterococcus faecalis strain, the attenuators fused with lacZ were cloned into the shuttle plasmid pJIM2246 and the plasmid was introduced into Staphylococcus aureus RN4220. For the wild-type attenuators, HMR 3004 was a very weak inducer, unlike its cladinose counterpart RU 6652 and erythromycin. As expected, for the fusion originating from the constitutively resistant S. agalactiae strain, the level of uninduced beta-galactosidase synthesis was high. For one S. pneumoniae attenuator, mutations in the 3' end of the attenuator that weakened the stem-loop structure that sequesters the ribosome-binding site and start codon for ermAM methylase could explain the high level of uninduced beta-galactosidase produced. For streptococci, the activity of HMR 3004 correlated with the basal level of beta-galactosidase synthesized. The weak inducer activity of HMR 3004 explained its activity against inducibly MLSB-resistant S. pneumoniae but did not correlate with the moderate activity of the antibiotic against inducibly resistant E. faecalis.
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PMID:A new ketolide, HMR 3004, active against streptococci inducibly resistant to erythromycin. 962 82

We have shown that the DNA demethylation complex isolated from chicken embryos has a G(.)T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a beta-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A (an analogue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction. Demethylation occurs downstream of the hormone response elements. No genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor alpha. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.
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PMID:Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene. 1129 68

One of the urgent tasks in understanding endocrine disruptors (EDs) is to compile a list of suspected substances among the huge number of chemicals by using the screening test method. An in vitro screening test is a more useful tool for primary selection of suspected EDs. We have developed an assay for EDs using the yeast two-hybrid system. The assay is based on the ligand-dependent interaction of two proteins, a hormone receptor and a coactivator, and the hormonal activity is detected by beta-galactosidase activity. This assay is a very simple and inexpensive test method with high repeatability to detect the agonist, and it is applicable for the detection of antagonist and active compounds after metabolism. Accordingly, it has been used in more than 40 laboratories in Japan. To date, we have tested the estrogenic activity of more than 500 chemicals including natural substances, medicines, pesticides and industrial chemicals. Sixty-four compounds were evaluated as positive and most of these possessed a common structure: phenol with a hydrophobic moiety at the para-position without bulky groups at the ortho-position. These results are expected to facilitate further risk assessment of chemicals, especially EDs.
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PMID:[Bioassay for endocrine disruptors by using yeast two-hybrid system]. 1157 61

Retinoid X receptor alpha (RXR alpha) is a member of the steroid hormone receptor superfamily. Using yeast two-hybrid screening, beta-galactosidase assays, and pull-down assays, we show that RNF8, a RING finger protein recently isolated as a protein binding to a ubiquitin-conjugating enzyme, binds to RXR alpha through the N-terminal regions of both proteins. In COS7 cells, overexpressed RNF8 colocalized and interacted with RXR alpha in the nucleus, as shown by fluorescence resonance energy transfer. A point mutation of RNF8, Cys-403 to Ser (C403S), which disrupts the RING finger structure, or deletion of the N-terminal region (Delta N) of RNF8 prevented localization of RNF8 to the nucleus without affecting nuclear localization of RXR alpha. Although transient overexpression of RNF8 had little effect on RXR alpha ubiquitination, RNF8 dose-dependently enhanced RXR alpha-mediated transactivation of the RXR-responsive element (RXRE)-bearing gene promoter without the addition of its ligand, 9-cis-retinoic acid (RA), and up-regulated the expression of the genes downstream of RXRE as well as an RA-response element. This transactivation-enhancing activity was not seen with either the C403S point mutant or the Delta N deletion mutant of RNF8. These results suggest a novel function of RNF8 as a regulator of RXR alpha-mediated transcriptional activity through interaction between their respective N-terminal regions.
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PMID:The RING finger protein, RNF8, interacts with retinoid X receptor alpha and enhances its transcription-stimulating activity. 1498 Oct 89

Dicofol is a non-systemic acaricide/miticide currently registered in the US and Canada for use on a wide variety of crops. This agrochemical has been identified as a potential candidate substance for the United Nations Economic Commission for Europe (UN-ECE) Persistent Organic Pollutant (POP) Protocol and implicated as a potential "endocrine disrupting compound". The technical product is usually synthesized from technical DDT and consists of approximately 80% and 20% of p,p'- and o,p'-dicofol isomers. The o,p'-substituted isomer of dicofol is chiral and may have enantiomer-specific activity; however, the stereospecific activity of o,p'-dicofol has not been reported. In this study, we examined the isomer- and enantiomer-specific endocrine disruption potential of dicofol using yeast-based steroid hormone receptor gene transcription assay designed with the human estrogen receptor (hER). Estrogenic activity of (+)-17-beta estradiol (positive control), p,p'-dicofol, racemic o,p'-dicofol [(+/-)-o,p'-dicofol] and the individual o,p'-dicofol enantiomers was measured via quantification of beta-galactosidase. The (+/-)-o,p'- and p,p'-dicofol were weak estrogen mimics (EC(50): 4.2 x 10(-6) and 1.6 x 10(-6)M, respectively) relative to estradiol (3.7 x 10(-10)M). For o,p'-dicofol, the beta-galactosidase induction by (-)-o,p'-dicofol (EC(50): 5.1 x 10(-7)M) was greater than the racemic mixture. However, the (+)-o,p'-dicofol enantiomer was found to have negligible estrogenic activity. These data indicate that dicofol is a weak hER agonist due to activity of the achiral p,p'-isomer and (-)-o,p'-substituted enantiomer and emphasizes the influence of chemical structure and configuration on biological responses to exposure from chiral compounds.
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PMID:Estrogenic activity of dicofol with the human estrogen receptor: Isomer- and enantiomer-specific implications. 1633 70

Steroid hormone receptor-mediated reporter assays in the budding yeast Saccharomyces cerevisiae have been an invaluable tool for the identification and functional characterization of steroid hormone receptor-associated chaperones and cochaperones. This chapter describes a hormone-inducible androgen receptor-mediated beta-galactosidase reporter assay in yeast. In addition, the immunophilin FKBP52 is used as a specific example of a receptor-associated cochaperone that acts as a positive regulator of receptor function. With the right combination of receptor and cochaperone expression plasmids, reporter plasmid, and ligand, the assay protocol described here could be used to functionally characterize a wide variety of nuclear receptor-cochaperone interactions. In addition to the functional characterization of receptor regulatory proteins, a modified version of this assay is currently being used to screen compound libraries for selective FKBP52 inhibitors that represent attractive therapeutic candidates for the treatment of steroid hormone receptor-associated diseases.
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PMID:Yeast-based reporter assays for the functional characterization of cochaperone interactions with steroid hormone receptors. 1911 43


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