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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cell line which can be used in a simple, sensitive, and rapid histochemical assay was isolated for detection of herpes simplex virus (HSV). The cell line was derived by selection of G418 resistant colonies following co-transfection of baby hamster kidney cells with a plasmid which contains a G418 antibiotic resistance marker and a plasmid which contains the Escherichia coli LacZ gene placed behind an inducible HSV promoter. The promoter is from HSV-1UL39 which encodes ICP6, the large subunit of ribonucleotide reductase (RR1). This promoter has a number of features which make it ideal for the detection of HSV. First, there is no constitutive expression from this promoter in uninfected cells. Second, activation of the promoter appears to be specific for HSV. Third, expression from this promoter occurs within hours after infection. Fourth, this promoter is strongly transactivated by the virion associated trans-activator protein VP16. As early as six hours after infection HSV-infected cells can be detected by histochemical staining for beta-galactosidase activity. Infected cells stain intensely blue whereas uninfected cells show no staining, and a single infected cell can easily be recognized in a microscopic field of uninfected cells. Both HSV-1 and HSV-2 are detected with this cell line, but after infection with human cytomegalovirus (HCMV), varicella zoster virus (VZV), adenovirus, and sindbis virus no blue cells were detected. Quantitation of HSV-1 stocks on this cell line by counting blue cell forming units (BFU) reveals that the number of BFU/ml closely approximates the number of plaque forming units (PFU)/ml as determined by plaque assays on the parent cell line. This cell line should provide a useful adjunct in the diagnostic virology laboratory for the rapid detection of HSV in clinical specimens.
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PMID:Isolation of a cell line for rapid and sensitive histochemical assay for the detection of herpes simplex virus. 132 70

The expression of the genes encoding ribonucleotide reductase in Escherichia coli was investigated in cultures synchronized by obtaining the smallest cells in a population after sucrose gradient centrifugation. Specific activity of ribonucleotide reductase and DNA initiation were found to increase in parallel, periodically as a function of the cell cycle. The expression of nrd was also determined in cells synchronized by periodic repeated doubling in a phosphate limited medium. Antibodies directed against the B2 subunit of ribonucleotide reductase were raised in a rabbit and purified. Immunoprecipitation of the B2 subunit and RNA-DNA dot blot hybridization assays were developed and employed to determine the expression of ribonucleotide reductase translational and transcriptional products during the cell cycle. Both of nrd-mRNA and B2 subunit expression were found to increase each generation at approximately the same time DNA synthesis was initiated and then to decrease back to the basal level shortly after DNA initiation. These results provided evidence of cell cycle dependent regulation of ribonucleotide reductase in E. coli. When the upstream regulatory region of nrd was fused to a promoterless lacZ gene on a single copy plasmid, lac-mRNA and beta-galactosidase were found to be synthesized in parallel to nrd expression from the chromosomal operon. When nrd sequences surrounding the promoter were removed from this construct, lac-mRNA and beta-galactosidase synthesis were no longer cell cycle regulated.
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PMID:Escherichia coli ribonucleotide reductase expression is cell cycle regulated. 138 14

Herpes simplex virus (HSV) encodes a ribonucleotide reductase consisting of two subunits (140 and 38 kilodaltons) whose genes map to coordinates 0.56 to 0.60 on the viral genome. Host cell lines containing the HpaI F fragment which includes the reductase subunit genes of HSV type 1 strain KOS (coordinates 0.535 to 0.620) were generated. Transfection of these cells with a plasmid containing the immediate-early ICP0 gene resulted in the expression of ICP6; interestingly, ICP4 plasmids failed to induce expression, indicating an unusual pattern of ICP6 regulation. One such cell line (D14) was used to isolate a mutant with the structural gene of lacZ inserted into the ICP6 gene such that the lacZ gene is read in frame with the N-terminal region of ICP6. This mutant generated a protein containing 434 amino acids (38%) of the N terminus of ICP6 fused to beta-galactosidase under control of the endogenous ICP6 promoter. Screening for virus recombinants was greatly facilitated by staining virus plaques with 5-bromo-4-chloro-3-indoyl-beta-D-galactoside (X-gal). Enzyme assays of infected BHK cells indicated that the mutant is incapable of inducing viral ribonucleotide reductase activity. Surprisingly, although plaque size was greatly reduced, mutant virus yield was reduced only four- to fivefold compared with that of the wild type grown in exponentially growing Vero cells. Mutant virus plaque size, yields, and ability to synthesize viral DNA were more severely compromised in serum-starved cells as compared with the wild type grown under the same condition. Although our evidence suggests that the HSV type 1 ribonucleotide reductase is not required for virus growth and DNA replication in dividing cells, it may be required for growth in nondividing cells.
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PMID:Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant. 282 47

Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.
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PMID:Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain. 810 47

The expression of the nrd operon encoding ribonucleotide reductase in Escherichia coli has been shown to be cell cycle regulated. To identify the cis-acting elements required for the cell cycle regulation of the nrd promoter, different 5' deletions as well as site-directed mutations were translationally fused to a lacZ reporter gene. The expression of beta-galactosidase from these nrd-lacZ fusions in single-copy plasmids was determined with synchronously growing cultures obtained by repeated phosphate starvation as well as with exponentially growing cultures by flow cytometry analysis. Although Fis and DnaA, two regulatory proteins that bind at multiple sites on the E. coli chromosome, have been found to regulate the nrd promoter, the results in this study demonstrated that neither Fis nor DnaA was required for nrd cell cycle regulation. A cis-acting upstream AT-rich sequence was found to be required for the cell cycle regulation. This sequence could be replaced by a different sequence that maintained the AT richness. A flow cytometry analysis that combined specific immunofluorescent staining of beta-galactosidase with a DNA-specific stain was developed and employed to study the nrd promoter activity in cells at specific cell cycle positions. The results of the flow cytometry analysis confirmed the results obtained from studies with synchronized cells.
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PMID:Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence. 815 11

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)
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PMID:The RR1 gene of herpes simplex virus type 1 is uniquely trans activated by ICP0 during infection. 839 74

A Mycoplasma hyopneumoniae clone bank was screened with hyperimmune pig serum. One clone exhibited sequence homology to the prokaryotic R2 subunit of ribonucleotide reductase and was expressed as an 11-kDa protein fused to beta-galactosidase. The vaccine potential of the fusion protein was assessed in pig trials. Following experimental challenge with a virulent isolate of M. hyopneumoniae, gross lung pathology (mean Goodwin lung score) of vaccinated animals, irrespective of adjuvant treatment, was significantly reduced compared with that of control unvaccinated pigs (P < 0.05).
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PMID:Molecular characterization of a ribonucleotide reductase (nrdF) gene fragment of Mycoplasma hyopneumoniae and assessment of the recombinant product as an experimental vaccine for enzootic pneumonia. 864 61

We have isolated the gene. rnrB, that encodes the ribonucleotide reductase small subunit of Dictyostelium discoideum. The deduced amino acid sequence of rnrB exhibits about 60% sequence identity with its homologues in other eukaryotes. As demonstrated by RNA blot analysis the rnrB transcript is detected in growing cells and decreases dramatically at the onset of development. The rnrB transcript reappears after the cells have formed multicellular aggregates. To further examine the pattern of expression, we have fused the rnrB promoter and part of its coding sequence to lacZ. The transgenic strain bearing such a reporter construct expresses the fusion gene with a biphasic profile, which is indistinguishable from that of the endogenous rnrB. The multicellular aggregates of Dictyostelium are differentiated along the anterior-posterior axis. Cells in the anterior give rise to the stalk of the fruiting body while cells in the posterior are precursors of spores. Results from histochemical staining show that beta-galactosidase activity is detected exclusively in the posterior two-thirds of the aggregates. These data suggest that rnrB is expressed in prespore cells during postaggregative development and in vegetative cells.
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PMID:A prespore-specific gene of Dictyostelium discoideum encodes the small subunit of ribonucleotide reductase. 895 Jan 85

We have examined the promoter of rnrB, the gene encoding the small subunit of ribonucleotide reductase of Dictyostelium discoideum, using lacZ as a reporter gene. Deletion analysis showed that expression of this gene in vegetative cells involves an A/T-rich element, whereas its expression in prespore cells during development requires a region encompassing two G/C-rich elements, designated box A and box B. Removal of boxes A and B results in very low level of activity. When either box A or box B is deleted, prestalk cells adjacent to the prespore zone also express beta-galactosidase. The behavior of these cis-regulatory elements implies that the mechanism regulating the prespore-specific expression of rnrB is different from that regulating other known prespore genes. We have used electrophoretic mobility shift assays to identify factors that interact with box A and box B. Box A interacts with a factor that is found in the nuclear fraction. While box B interacts with a factor that is present in the cytosolic fraction throughout growth and development, its presence in the nuclear fraction is developmentally regulated. Results from competition assays suggest that both box A and box B interact with transcriptional activators that have not been characterized previously.
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PMID:Identification of cis-regulating elements and trans-acting factors regulating the expression of the gene encoding the small subunit of ribonucleotide reductase in Dictyostelium discoideum. 1040 Jun 62

We evaluated the feasibility of delivering a gene into monkey eyes using a replication-competent herpes simplex virus (HSV) type 1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene. To determine the efficiency of in vitro HSV-mediated gene transfer, cultured human trabecular meshwork (HTM) and human ciliary muscle (HCM) cells were infected with hrR3 and beta-galactosidase activity was measured histochemically. Six cynomolgus monkey eyes received viral injections into the anterior chamber (2 x 10(7) pfu) and/or the vitreous (5 x 10(7) pfu), and the distribution of cells expressing lacZ was evaluated. In vitro, both cultured HTM and HCM cells displayed multiplicity-dependent beta-galactosidase activity. In vivo, intracameral and/or intravitreal injection resulted in transgene expression in TM cells and in non-pigmented ciliary epithelial cells (NPE), but not in CM cells. Transgene expression was also detected in retinal pigmented epithelial (RPE) cells and sporadic retinal ganglion cells (RGC) in eyes receiving virus intracamerally and intravitreally respectively. We observed significant inflammation in the anterior chamber, TM and CM in virus-injected eyes, along with mild vitritis and retinitis. This study demonstrates successful gene transfer using hrR3 as a vector in human ocular cells and in ocular tissues in living monkeys. Further investigation of the etiology of the inflammatory response, possible cytotoxicity, and limited duration of transgene expression is necessary in order to make this technique clinically applicable.
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PMID:Herpes simplex virus mediated gene transfer to primate ocular tissues. 1050 72


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