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)

Treatment of Escherichia coli with DNA-damaging agents results in the increased expression of a set of din (damage-inducible) genes. We have studied the regulation and function of these genes by using the Mud(Ap, lac) bacteriophage to obtain fusions of the beta-galactosidase structural gene to the promoters of various din genes. By this technique, we have shown that the uvrA, uvrB, and umuC genes are induced by UV and other DNA damaging agents. The products of the uvrA and uvrB genes are required for the excision repair of pyrimidine dimers and other bulky lesions; the umuC gene product is required for most chemical mutagenesis in E. coli. Genetic analyses of all of the din-lac fusions isolated to date indicate that lexA is the direct repressor of each of the din genes and that proteolytic cleavage of the lexA protein is required for their induction. We have also been studying the mechanism by which the clinically isolated plasmid pKM101 increases the susceptibility of cells to chemical mutagenesis. Inasmuch as the effects of pKM101 on mutagenesis are recA+ lexA+ -dependent and the plasmid can suppress the nonmutability of a umuC mutant, it seems likely that pKM101 may carry an analog of the chromosomal umuC gene. By insertion mutagenesis using Tn5, we identified an approximately 2,000-base pair region of pKM101 which is necessary for its effects on mutagenesis.
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PMID:Mutagenesis and cellular responses to DNA damage. 681 2

An antibody specific for puromycin (PU) was prepared by immunization of rabbits with a PU conjugate of bovine serum albumin, which was newly synthesized by coupling PU to mercaptosuccinylated bovine serum albumin via a cross-linker, N-(gamma-maleimidobutyryloxy)succinimide. Enzyme labeling of PU was performed using beta-D-galactosidase [EC 3.2.1.23] via N-(m-maleimidobenzoyloxy) succinimide. An ultrasensitive and specific enzyme immunoassay for PU was developed utilizing these reagents by a double antibody technique. The standard curve of the assay was linear in the range of 2 pg to 100 pg, and the lower limit of detection was 28.2 pm (2 pg/tube); so the enzyme immunoassay was found to be approximately 326,000 times more sensitive than a microbiological assay. Further, the enzyme immunoassay is free from interference by any purine or pyrimidine analogs, or by other drugs commonly used for the inhibition of protein synthesis. Using this assay, drug levels were easily determined in rat tissue following PU administration. Since PU is extensively available as an inhibitor of protein synthesis, the enzyme immunoassay should provide useful tool for developing biochemical and toxicity studies of PU.
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PMID:The preparation of puromycin antibody and its use in enzyme immunoassay for the quantification using beta-D-galactosidase as a label. 681 27

A purE::lac fusion strain was isolated by using a special Mu phage developed by M. Casadaban. In the presence of adenine (100 micrograms/ml), beta-galactosidase synthesis was repressed by greater than 90%. beta-Galactosidase activity could be detected 6 to 8 min after the removal of adenine and increased linearly for at least 20 min. purR- mutants were isolated and synthesized 1.7- to 1.8-fold-higher levels of beta-galactosidase compared with purR+ cells. Azaserine derepressed purE transcription approximately 1.7-fold by lowering purine nucleotide pools. Glutamine and pyrimidine supplementation or starvation had no effect on purE transcription. A comparison of the rate of de novo purine biosynthesis and purE transcription indicated that the in vivo rate of de novo purine biosynthesis was more sensitive to the inhibitory effects of adenine than was transcription at the purE locus.
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PMID:Regulation of purE transcription in a purE::lac fusion strain of Escherichia coli. 703 38

The effect of CH-123 (3-carbethoxy-6-methyl-1-9-(carboxy-methyl)-1-4-oxo-6,7,8,9-tetrahydro-4H-pyrid o(1,2a)pyrimidine) was investigated on the activity of 4 lysosomal enzymes: beta-glucuronidase, beta-galactosidase, N-acetyl-beta-glucosaminidase and acid phosphatase obtained from aortic smooth muscle and liver cells of rabbits. Animals were fed on a 2% cholesterol diet for 4 weeks and used an experimental atherosclerotic group. In drug-treated groups, after 4 weeks of cholesterol feeding the diet was changed to regular food and the animals were treated daily either with 50 mg/kg CH-123 or with 250 mg/kg Clofibrate. The postnuclear supernatant of homogenates of liver and aortic cells was isolated, lysosomes were fractionated by sucrose density gradient centrifugation, and the activity of enzymes was measured. In cholesterol-fed animals the enzyme activities of aorta and liver was 3-5 times higher than in the control, i.e. in the group of rabbits fed regular food. On Clofibrate treatment the enzyme activities were 2-3 times higher, but on treatment with CH-123, they were only 1.2-1.8 times above the control. Experiments suggest that CH-123 treatment suppresses the elevated lysosomal marker enzyme activities in aortic and liver cells of atherosclerotic animals.
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PMID:Effect of CH-1243, a pyrido (1,2-a) pyrimidine derivative on the elevated activity of lysosomal enzymes of rabbit aorta and liver in experimental atherosclerosis. 724 98

The relationship between the induction of the genes RAD54 and RNR2 and the induction and repair of specific DNA lesions was studied in the yeast Saccharomyces cerevisiae using Rad54-lacZ and RNR2-lacZ fusion strains. Gene induction was followed by measuring beta-galactosidase activity. At comparable levels of furocoumarin-DNA photoadducts, RAD54 was more effectively induced by bifunctional than by monofunctional furocoumarins indicating that mixtures of monoadducts (MA) and interstrand cross-links (CL) provide a stronger inducing signal than MA. RNR2 induction kinetics were measured in relation to cell growth and survival responses after treatment with the furocoumarins 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), 3-carbethoxypsoralen (3-CPs), 7-methyl-pyrido[3,4-c]psoralen (MePyPs) and 4,4',6-trimethylangelicin (TMA), benzo[a]pyrene (B(a)P and 1,6-dioxapyrene (1,6-DP) plus UVA, 254 nm UV radiation and cobalt-60 gamma-radiation. Induction of RNR2 took place during the DNA repair period before resumption of cell growth and clearly increased with increasing equitoxic dose levels. Treatments with furocoumarin plus 365 nm radiation (UVA) and 254 nm (UV) radiation were effective inducers whereas gene induction was relatively weak after gamma-radiation and absent after the induction of oxidative damage by B(a)P and 1,6-DP and UVA. The results suggest that it is the specific processing of different DNA lesions that determines the potency of the induction signal. Apparently, DNA lesions such as CL, and probably also closely located MA or pyrimidine dimers in opposite DNA strands involving the formation of double-strand breaks as repair intermediates, are most effective inducers.
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PMID:Induction of the genes RAD54 and RNR2 by various DNA damaging agents in Saccharomyces cerevisiae. 752 Sep 95

The preparation of a series of quinazoline-2,4-diones, 1-3, and pyrrolo[3,4-d]pyrimidine-2,4-diones, 4-8 is described. A small number of quinazolinedione analogs were identified from random screening to possess low micromolar (1.3-4.4 microM) potency in the nuclear factor of activated T cells-1-regulated beta-galactosidase expression assay. An expanded analog search resulted in identifying pyrrolopyrimidinedione 4b which is 5-10-fold (0.26 microM) more potent than the quinazolinediones. Replacement of the benzyl group with naphthyl led to greater potency and conformationally restricted analogs 4u-w. The naphthyl and acenaphthyl analogs are 10-100 times more potent inhibitors of beta-galactosidase expression than 4b. Binding affinity data for displacement of radiolabeled 4s from Jurkat cell membranes reflected an excellent correlation with the IC50 value for inhibition of beta-galactosidase activity. These products, whose structure-activity relationships are discussed, are of interest as potential agents for preventing interleukin-2 gene transcription.
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PMID:Novel inhibitors of the nuclear factor of activated T cells (NFAT)-mediated transcription of beta-galactosidase: potential immunosuppressive and antiinflammatory agents. 762 96

The mouse homeodomain protein Hoxa-7, expressed under the control of an inducible promoter, was able to inducibly activate reporter genes containing multimerized Hoxa-7 binding sites in Saccharomyces cerevisiae. This tight regulation was exploited in an attempt to screen for Hoxa-7 responsive elements. A reporter library consisting of a randomised 10 bp element inserted into the minimal gal1 promoter was constructed. In a surprisingly small screen, 24 reporters were isolated which had all of the transactivation characteristics expected for a Hoxa-7 binding site insertion. However, further characterisation revealed that the selected elements lacked homeodomain (HD) binding core motifs and were not bound by a purified Hoxa-7/beta-galactosidase fusion protein capable of binding known sites. The minimal promoter context contains 16 HD core motifs in 410 bp. Careful re-examination of basal levels revealed a low residual response of the gal1 minimal promoter to Hoxa-7. The 11 characterised 10 bp inserts amplified Hoxa-7 responsiveness in a manner correlated to increases in basal reporter activity. Thus, a quantitative range of Hox-responsiveness was produced by slight sequence alterations that did not change HD binding sites of their relative spacing in the promoter. These data suggest how, without altering resident HD base contact zones, mouse promoters could be optimised by natural selection to give appropriate quantitative outputs in each anatomical region defined by an assortment of Hox proteins. The selected elements were pyrimidine rich on the sense strand, containing (T)nC motifs, strikingly similar to sequences which enhance Hoxa-7 binding and activation from outside the HD contact zone. A search of defined sequence databases demonstrated that these elements were over-represented in promoters. Two elements altered the mobility shift patterns produced by cell extracts on minimal promoter fragments.
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PMID:Selection of ten base-pair sequences which enhance the response of the Gal1 minimal promoter to murine Hoxa-7 in yeast. 770 67

Expression of the Bacillus subtilis pyr operon is regulated by exogenous pyrimidines and the protein product of the first gene of the operon, PyrR. It has been proposed that PyrR mediates transcriptional attenuation at three untranslated segments of the operon (R.J. Turner, Y. Lu, and R.L. Switzer, J. Bacteriol., 176:3708-3722, 1994). In this study, transcriptional fusions of the pyr promoter followed by the pyr attenuation sequences, either individually or in tandem to a lacZ reporter gene, were used to examine the physiological functions of all three attenuators through their ability to affect beta-galactosidase expression. These fusions were studied as chromosomal integrants in various B. subtilis strains to examine the entire range of control by pyrimidines, PyrR dependence, amd developmental control of pyr gene expression. The nutritional regulation of each attenuator separately was roughly equivalent to that of the other two and was totally dependent upon PyrR, and that of tandem attenuators was cumulative. The regulation of a fusion of the spac promoter followed by the pyrP:pyrB intercistronic region to lacZ produced results similar to those obtained with the corresponding fusion containing the pyr promoter, demonstrating that attenuator-dependent regulation is independent of the promoter. Extreme pyrimidine starvation gave rise to two- to threefold-higher levels of expression of a pyr-lacZ fusion that lacked attenuators, independent of PyrR, than were obtained with cells that were not starved. Increased expression of a similar spac-lacZ fusion during pyrimidine starvation was also observed, however, indicating that attenuator-independent regulation is not a specific property of the pyr operon. Conversion of the initiator AUG codon in a small open reading frame in the pyrP:pyrB intercistronic region to UAG reduced expression by about half but did not alter regulation by pyrimidines, which excludes the possibility of a coupled transcription-translation attenuation mechanism. Developmental regulation of pyr expression during early stationary phase was found to be dependent upon the attenuators and PyrR, and the participation of SpoOA was excluded.
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PMID:Roles of the three transcriptional attenuators of the Bacillus subtilis pyrimidine biosynthetic operon in the regulation of its expression. 786 7

The sequence of the Penicillium chrysogenum pgkA gene promoter was determined up to 952 nucleotides (nt) 5' to the major transcriptional start point (position +1), and contains a 38 bp pyrimidine-rich region within which transcription initiates at this and two minor sites (-11, -23). A 21 bp segment (-99 to -79) closely matches a region which is essential for the expression of the Aspergillus nidulans pgkA gene. A further region was found with similarity to sequences in other A. nidulans promoters possibly effecting response to carbon source. The terminator region of the P. chrysogenum pgkA gene was sequenced as far as 192 nt 3' to the stop codon and three polyadenylation sites were found at 94, 103 and 107 nt from this point, the first preceded by a possible polyadenylation signal. No transcription termination signal was found but several regions potentially forming stem-loop-structures were noted. A single 1.3 kb pgkA mRNA was readily detected by Northern blot analysis of total cellular RNA. Steady-state levels of pgkA mRNA were 1.5 to 2.0 times greater in mycelium harvested at similar stages of growth from medium containing the carbon sources acetate or quinate compared to glucose. A transformed strain of P. chrysogenum containing a fusion of the pgkA promoter to the Escherichia coli lacZ reporter gene integrated at the oliC locus was constructed, and beta-galactosidase activity monitored during growth of batch cultures in defined media. The pgkA promoter activity increased during exponential growth and was 2-3 times greater and increased most rapidly in mycelium grown on quinate or acetate compared to glucose.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of the 3-phosphoglycerate kinase gene (pgkA) of Penicillium chrysogenum. 819 80

The effect of UV photoproducts or benzo[a]pyrene-diol-epoxide-I (BPDE-I) adducts in DNA on the transient expression of a reporter gene was measured in mammalian cells. The plasmid pRSVCAT was UV irradiated or treated with BPDE-I in vitro and co-transfected with undamaged pRSVBGAL into mouse and human fibroblasts. Variations in transfection efficiency among different cell lines were corrected by adjusting the volumes of cell extracts used in the chloramphenicol acetyl transferase (CAT) assays to contain equal beta-galactosidase (BGAL) activity. The expression of the CAT gene was found to decrease exponentially after transfection of pRSVCAT containing increasing numbers of DNA lesions per molecule. The average number of BPDE-I adducts per plasmid molecule was measured by ELISA; the average number of pyrimidine dimers was estimated from the dose kinetics for the disappearance of the supercoiled form of irradiated plasmid DNA treated with Micrococcus luteus UV endonuclease. By expressing the inhibition of CAT activity in terms of the average number of lesions per gene, we were able to compare directly the effects of two different carcinogen lesions on transient transcription. We observed comparable kinetics of inhibition of gene expression by BPDE-I adducts and pyrimidine dimers in DNA. D0 values determined by linear regression analysis of dose-response curves for inhibition of CAT activity were 4.9 BPDE-I adducts or 6.6 pyrimidine dimers per gene in excision-proficient human fibroblasts; the corresponding values in mouse cells were 4.4 BPDE-I adducts or 5.5 pyrimidine dimers. Similar threshold densities of BPDE-I adducts and pyrimidine dimers were observed before inhibition of transcription from pRSVCAT was detected. No threshold was observed in experiments with human fibroblasts deficient in excision repair (xeroderma pigmentosum group A); calculated D0 values were 1.2 pyrimidine dimers of 2.1 BPDE-I adducts. Our results permit direct comparisons of the magnitude of inhibition of gene transcription by distinct DNA lesions, and suggest that BPDE-I adducts and UV-induced cyclobutane pyrimidine dimers in template DNA block transcription with similar efficacy.
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PMID:Inhibition of reporter gene expression in mammalian cells. Effects of distinct carcinogen lesions in DNA. 820 75


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