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:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The US6 gene family, located within the unique short region (US) of the human cytomegalovirus (HCMV) genome, contains six open reading frames (US6 through US11) which may encode glycoproteins, such as gcII (D. Gretch, B. Kari, R. Gehrz, and M. Stinski, J. Virol. 62:1956-1962, 1988). By homologous recombination, several different recombinant HCMV were created which contain a marker gene, beta-glucuronidase, inserted within this gene family. It was demonstrated that beta-glucuronidase has utility as a marker gene for the identification of recombinants in this herpesvirus system, without the occurrence of deletions in other regions of the viral genome. DNA and RNA blot analyses attested to the fidelity of the recombination. Immunoprecipitation experiments using monospecific polyclonal antisera indicated that the US10 and/or US11 gene products were not expressed in the recombinants, as predicted. These results, along with single-cycle growth analyses, indicated that the US10 and US11 gene products are nonessential for virus replication and growth in tissue culture. HCMV recombinants expressing beta-glucuronidase seemed to be genetically stable.
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PMID:Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential. 165 74

The tetracycline repressor (TetR)-regulated gene expression system from Escherichia coli was used to control gene expression in recombinant human cytomegalovirus (HCMV). To adapt the TetR system in HCMV, derivatives of the viral US11 (early) gene promoter, which controls the beta-glucuronidase reporter gene, were constructed by systematic insertion of the tetracycline operator (tetO) sequences. Gene expression from constructs containing two or three appropriately placed tetO sequences adjacent to the TATA box were efficiently repressed by a TetR-VP16 fusion protein (tTA) in a transient expression system. Efficient repression (50- to 120-fold) also occurred in tTA-expressing stably transfected human cells which were infected with recombinant HCMV containing a US11 promoter surrounded by three tetO sequences. The tTA-mediated gene repression was relieved in the presence of 1 microgram of tetracycline per ml. The results of this study are significant in three respects. (i) This is the first demonstration that a TetR-derived protein can be used to efficiently repress gene expression in a mammalian system. (ii) Efficient repression was dependent on the presence of the transcriptional activation domain from the herpes simplex virus type 1 VP16 protein. (iii) The ability to regulate gene expression in a controlled fashion in order to elucidate viral gene function is an important development in the HCMV field. The tTA-mediated gene repression system may be extremely useful for creating host-range mutants in essential genes in order to study their role in the HCMV replicative cycle, a system that is otherwise exceedingly difficult to genetically dissect.
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PMID:Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus. 788 7

We have developed a system to study human cytomegalovirus (HCMV) cis-acting promoter elements within the context of the viral genome. A recombinant HCMV (RV134) containing a marker gene (beta-glucuronidase) was used to insert HCMV promoter-chloramphenicol acetyltransferase gene constructs into the viral genome between open reading frames US9 and US10. Using this system, we have studied the promoters for the early DNA polymerase gene (UL54), the early-late lower matrix phosphoprotein gene (pp65, UL83), and the true late 28-kDa structural phosphoprotein gene (pp28, UL99). Transient-expression assays demonstrated that the pp65 and pp28 promoters are activated earlier and to higher levels than typically observed with the endogenous gene. In contrast, insertion of these promoters into the viral genome resulted in kinetics which mimicked that of the endogenous genes. In addition, we have also tested a variant of the pp28 promoter (d24/26CAT) which is deleted from -609 to -41. This promoter behaved similarly to the wild-type pp28 promoter, indicating that sequences from -40 to +106 are sufficient for conferring true late kinetics. Taken together, these data demonstrate that the viral genome affords a level of regulation on HCMV gene expression that has been previously unrealized. Therefore, these experiments provide a model system for the analysis of cis-acting promoter regulatory elements in the context of the viral genome.
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PMID:Use of recombinant virus to assess human cytomegalovirus early and late promoters in the context of the viral genome. 808 94

We report the construction and expression of a fusion protein between a single-chain antibody specific for human carcinomas and human beta-glucuronidase by recombinant DNA technology. The sequences encoding the murine monoclonal antibody 323/A3 light- and heavy-chain variable genes were joined by a synthetic sequence encoding a 15-amino-acid linker and combined with human beta-glucuronidase by a synthetic sequence encoding a 6-amino-acid linker. The construct was placed under the control of the cytomegalovirus promotor and expressed in COS-7 cells. The yield of active fusion protein was 10 ng/ml transfectoma supernatant. Antibody affinity, antibody specificity and enzyme activity were fully retained by the fusion protein. Biochemical characterization of the fusion protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed a molecular mass of 100 kDa under denaturing conditions. Gel-filtration analysis indicated that the enzymatically active form is a tetramer of approximately 400 kDa. The non-toxic prodrug N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate was activated to the cytotoxic drug doxorubicin by the fusion protein with a hydrolysis rate similar to that of human beta-glucuronidase. The growth inhibition of tumor cells coated with the fusion protein and exposed to prodrug was similar to that obtained with doxorubicin. This study shows the feasibility of constructing eukaryotic fusion proteins consisting of a single-chain antibody and human beta-glucuronidase for use in the specific activation of anticancer prodrugs.
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PMID:Construction and characterization of a fusion protein of single-chain anti-carcinoma antibody 323/A3 and human beta-glucuronidase. 943 50

The central nervous system (CNS) is a predominant site of involvement in several lysosomal storage diseases (LSDs); and for many patients, these diseases are diagnosed only after the onset of symptoms related to the progressive accumulation of macromolecules within lysosomes. The mucopolysaccharidosis type VII (MPS VII) mice are deficient for the lysosomal enzyme beta-glucuronidase and, by early adulthood, develop a significant degree of glycosaminoglycan storage within neuronal, glial, and leptomeningeal cells. Using this animal model, we investigated whether gene transfer mediated by a recombinant adeno-associated virus (rAAV) vector is capable of reversing the progression of storage lesions within the CNS. Adult MPS VII mice received intracerebral injections of 4 X 10(7) infectious units of a rAAV vector carrying the murine beta-glucuronidase (gus-s(a)) cDNA under the transcriptional direction of the cytomegalovirus immediate-early promoter and enhancer. By 1 month after vector administration, transgene-derived beta-glucuronidase was present surrounding the injection site. Enzyme levels were between 50 and 240% of that found in wild-type mice. This level of beta-glucuronidase activity was sufficient to reduce the degree of lysosomal storage. Moreover, the reduction in storage was maintained for at least 3 months post-rAAV administration. These data demonstrate that rAAV vectors can transduce the diseased CNS of MPS VII mice and mediate levels of transgene expression necessary for a therapeutic response. Thus, rAAV vectors are potential tools in the treatment of the mucopolysaccharidoses and other lysosomal storage diseases.
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PMID:Recombinant adeno-associated virus-mediated correction of lysosomal storage within the central nervous system of the adult mucopolysaccharidosis type VII mouse. 1072 30

In 30 patients with mononucleosis-like syndrome (MLS) caused by cytomegalovirus (CMV), diagnosed on the basis of clinical symptoms, haematological & serological changes (after excluding Epstein-Barr virus, HAV, HBV and HCV infections), the following measurements were done weekly during consecutive two months': bilirubin concentration, aspartate & alanine aminotransferases (AST & ALT), alkaline phosphatase (ALP), beta-glucuronidase (B-GR), and gamma-glutamyltranspeptidase (GGTP) activity. Increase in bilirubin concentration was found in 6% of patients, increase of AST and ALT activity--in 70%, GGTP--in 50%, ALP--in 25%, and of B-GR--in 16% of the subjects. The highest bilirubin concentration, and high levels of AST, ALT, and B-GR were noted in the 2nd week of infection, whereas the peak activity of ALP and GGTP was found in the 3rd week of the disease. In all patients normalization of bilirubin concentration was earliest (5th week of infection); followed by decrease of AST, ALT, B-GR, and ALP activity (7th week), and subsequently--that of GGTP (8th week of the disease). The results of the investigations have shown that in the course of MLS the changes of hepatic activity are limited and transient; they return to normal synchronously with the withdrawal of clinical symptoms (4th-6th week of the disease), without permanent measurable consequences. In patients with MLS and increase AST & ALT activity (400-600 iu) as well as slight increased of bilirubin concentrations hepatitis C,A and B should be excluded. In has not been established so far whether the changes of hepatic function during MLS are the consequence of direct infection by CMV, reactivation of the primary occult infection (asymptomatic), or re-infection by a different serotype.
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PMID:[Biochemical changes of liver damage factors in the course of mononucleosis like syndrome caused by cytomegalovirus]. 1134 95

Conditions for the production of protoplasts and gene transfer in Pythium aphanidermatum were investigated. Efficient protoplast generation was possible after culture of mycelium in potato dextrose broth followed by digestion with 0.5% (w/v) each of cellulase and beta- d-glucanase. Plasmid pHAMT35N/SK encoding the nptII gene under control of the Ham34 promoter from the oomycete Bremia lactucae was used to define electroporation parameters for gene transfer. A square-wave electroporation pulse of 2500 V/cm at 50 microF capacitance reproducibly produced transformants, albeit at low efficiency (0.1-0.4 transformants from approximately 10(5) regenerable protoplasts per microgram of DNA). Thirty-two independent transformants exhibited wild-type growth on potato dextrose agar amended with geneticin at 50 microg/ml, a concentration that near completely inhibited the growth of untransformed P. aphanidermatum. Southern blot analysis indicated that transforming DNA was integrated into the oomycete genome and that the DNA was stably inherited through sporogenesis. Growth on geneticin-free media, the ability to form zoospores or oospores, and the ability to cause disease in sugarbeet seedlings in the laboratory were indistinguishable between a subset of the transformed isolates and the progenitor isolate 898B. Co-electroporation of pHAMT35N/SK with plasmid pACT-GUS encoding the Escherichia coli gusA gene controlled by oomycete transcriptional promoter and terminator sequences or with pEGFP encoding enhanced green fluorescent protein under the control of the immediate early promoter from the mammalian cytomegalovirus produced, respectively, stable beta-glucuronidase and transient expression of blue-green fluorescence. Application of the technique to studies on the biochemical basis for pathogenesis in this agriculturally important group of fungi is discussed.
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PMID:Transformation of Pythium aphanidermatum to geneticin resistance. 1261 8

To locate the transmissible gastroenteritis coronavirus (TGEV) packaging signal, the incorporation of TGEV subgenomic mRNAs (sgmRNAs) into virions was first addressed. TGEV virions were purified by three different techniques, including an immunopurification using an M protein-specific monoclonal antibody. Detection of sgmRNAs in virions by specific reverse transcription-PCRs (RT-PCRs) was related to the purity of virus preparations. Interestingly, virus mRNAs were detected in partially purified virus but not in virus immunopurified using stringent conditions. Analyses by quantitative RT-PCR confirmed that virus mRNAs were not present in highly purified preparations. Lack of sgmRNA encapsidation was probably due to the absence of a packaging signal (Psi) within these mRNAs. This information plus that from the encapsidation of a collection of TGEV-derived minigenomes suggested that Psi is located at the 5' end of the genome. To confirm that this was the case, a set of minigenomes was expressed that included an expression cassette for an mRNA including the beta-glucuronidase gene (GUS) plus variable sequence fragments from the 5' end of the virus genome potentially including Psi. Insertion of the first 649 nucleotides (nt) of the TGEV genome led to the specific encapsidation of the mRNA, indicating that a Psi was located within this region which was absent from all of the other virus mRNAs. The presence of this packaging signal was further confirmed by showing the expression and rescue of the mRNA including the first 649 nt of the TGEV genome under control of the cytomegalovirus promoter in TGEV-infected cells. This mRNA was successfully amplified and encapsidated, indicating that the first 649 nt of TGEV genome also contained the 5' cis-acting replication signals. The encapsidation efficiency of this mRNA was about 30-fold higher than the genome encapsidation efficiency, as estimated by quantitative RT-PCR. In contrast, viral mRNAs presented significantly lower encapsidation efficiencies (about 100-fold) than those of the virus genome, strongly suggesting that TGEV mRNAs in fact lacked an alternative TGEV Psi.
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PMID:Transmissible gastroenteritis coronavirus packaging signal is located at the 5' end of the virus genome. 1282 29

Cauliflower mosaic virus 35S promoter, widely used in transgenic crop plants, is known to be recognized in widely differing kinds of cells. Its activity in human cells may have impact on the risk assessment for the environmental release of genetically modified plants. In this study, transient expression of several constructs containing beta-glucuronidase (GUS) gene driven by cauliflower mosaic virus 35S promoter or by immediate early promoter of human cytomegalovirus (pCMV) was tested in both potato leaf protoplasts and cultured human cells. The results showed very low but measurable activity of 35S promoter in human 293T-cells (0.01% of that revealed when using pCMV) and in 293 cells that do not produce SV40 T antigen this activity was even lower. On the other hand, in potato protoplasts, pCMV displayed nearly 1% activity seen with p35S.
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PMID:Comparison of hCMV immediate early and CaMV 35S promoters in both plant and human cells. 1289 Jun 6

The 230-kbp murine cytomegalovirus (MCMV) genome is predicted to encode 182 open reading frames (orfs). One gene whose functional role is not known is encoded by the 762-bp m136 orf. Sequence analysis of rat cytomegalovirus (RCMV) strains Maastricht and English revealed homologous orfs, pr136, and ORF HJ4, respectively. Conservation of these orfs suggested that m136 and the RCMV homologs might play a role during virus replication. Expression of an epitope tagged form of m136 (m136-V5) yielded a polypeptide of 34 kDa that localized to the perinuclear region of transfected mouse 3T3 fibroblasts. Three independently generated MCMV m136 mutants were isolated and characterized. Mutations were introduced into the m136 orf by inserting either a beta-glucuronidase (m136-beta-gluc) or a guanosine phosphoribosyl transferase (m136-gpt) expression cassette into a unique BglII site, or by inserting a gpt cassette into a deleted region (Deltam136) of m136. No differences were observed in viral yield, plaque size, and plaque morphology between the parental strain and any of the m136 mutant viruses. In vivo analysis using a SCID mouse virulence model showed a consistently measurable attenuated phenotype for all three m136 mutants. The results showed that although the m136 gene was not essential for replication in vitro or in vivo, an intact m136 gene was necessary to yield wild type virulence during infection of the host.
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PMID:Characterization of the murine cytomegalovirus m136 gene. 1714 24


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