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 contribution of the herpes simplex virus type 1 (HSV-1)-encoded uracil DNA glycosylase (UNG), thymidine kinase (TK), and dUTPase to the relative mutant frequency (RMF) of the virus in cultured murine cells was examined. A panel of HSV-1 mutants that lacked singly or doubly the UNG, TK, or dUTPase activity were generated by disruption of the enzyme coding regions with the Escherichia coli beta-galactosidase (beta-gal) gene in strain 17syn+. To establish a baseline RMF of strain 17syn+, the beta-gal gene was inserted into the UL3 locus. In all of the viruses, the beta-gal insert served as a phenotypic marker of RMF. A mutant plaque was identified by the lack of beta-gal activity and, in selected cases, positive in situ hybridization for beta-gal sequences. Replication kinetics in NIH 3T3 cells demonstrated that all of the mutants replicated efficiently, generating stocks with equivalent titers. Two independently generated UL3-beta-gal viruses were examined and established a baseline RMF of approximately 0.5% in both NIH 3T3 and LM TK- cells. Loss of dUTPase activity resulted in viruses with fivefold-increased RMFs, indicating that the HSV-1 dUTPase has an antimutator function. The RMF observed for the tk- viruses was reduced as much as 40-fold (RMF of 0.02%), suggesting that the viral TK is a mutator activity. The RMF of two independent UNG- viruses showed no significant difference from the baseline RMF in limited passage; however, following successive passage, the data suggested that UNG activity serves as an antimutator. These results have implications for the natural history of HSV and the development of antiviral therapies.
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PMID:Mutations in accessory DNA replicating functions alter the relative mutation frequency of herpes simplex virus type 1 strains in cultured murine cells. 820 26

Transient expression assays with the herpes simplex virus type 1 (HSV-1) promoter/leader controlling the beta gamma (leaky-late) VP5 (UL19) mRNA encoding the major capsid protein showed that no more than 36 to 72 bases of VP5 leader are required for full-level expression. Constructs lacking the viral leader and the transcription initiation site expressed the reporter gene at about 20% of the maximum level. We confirmed this observation by using recombinant viruses in which VP5 promoter/leader deletions controlling the bacterial beta-galactosidase gene were inserted into the nonessential glycoprotein C (UL44) locus of the genome. Sequences within +36 are required for full-level expression, and removal of all leader sequences including the cap site resulted in a 10-fold decrease in reporter mRNA accumulation. The removal of the leader sequence had a measurable effect upon the kinetics of reporter mRNA accumulation, but insertion of the entire VP5 leader and cap site into a construct in which the reporter gene was controlled by the kinetically early (beta) dUTPase (UL50) promoter did not result in any significant change in the kinetics of dUTPase promoter expression. These results suggest that DNA sequences both 5' and 3' of the TATA box are important determinants of the beta gamma kinetics and levels of VP5 mRNA accumulation in the infected cell.
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PMID:Mutational analysis of sequences downstream of the TATA box of the herpes simplex virus type 1 major capsid protein (VP5/UL19) promoter. 839 39

Sequence analysis within the long segment of the pseudorabies virus (PrV) genome identified an open reading frame of 804 bp whose deduced protein product of 268 amino acids exhibited homology to dUTPases of other herpesviruses. The gene was designated UL50 because of its colinearity with the homologous gene of herpes simplex virus type 1. An antiserum raised against a bacterially expressed fragment of PrV UL50 specifically detected a 33-kDa protein in lysates of infected cells, which is in agreement with the predicted molecular mass of the PrV UL50 protein. A UL50-negative PrV mutant (PrV UL50-) was constructed by the insertion of a beta-galactosidase expression cassette into the UL50 coding sequence. A corresponding rescuant (PrV UL50resc) was also isolated. The interruption of the UL50 gene led to the disappearance of the 33-kDa protein, whereas restoration of UL50 gene expression restored detection of the 33-kDa protein. Enzyme activity assays confirmed that UL50 of PrV codes for a dUTPase which copurifies with nuclei of infected cells. PrV UL50- replicated with an only slightly reduced efficiency in epithelial cells in culture compared with that of its parental wild-type virus strain. Our results thus demonstrate that UL50 of PrV encodes a protein of 33 kDa with dUTPase activity which copurifies with nuclei of infected cells and is dispensable for replication in cultured epithelial cells.
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PMID:Identification and characterization of pseudorabies virus dUTPase. 855 88

Functional analysis of two promoters controlling early herpes simplex virus type 1 (HSV-1) transcripts encoding the UL37 and UL50 (dUTPase) proteins are described in this report. Transcripts expressed under the control of these promoters were found to be expressed early regardless of the position of the transcription unit within the viral genome. Despite this, wt dUTPase mRNA was 6-10 times more abundant than the UL37 transcript both in wt and recombinant viruses. This same difference in transcript abundance was seen when a reporter gene (beta-galactosidase) was controlled by the two promoters in recombinant viruses in the heterologous glycoprotein C (gC) locus. Thus, both the kinetics and relative abundance of UL50 and UL37 transcripts are a direct function of their respective promoter regulatory elements. Characterization of mutated UL37 and UL50 promoters in recombinant viruses showed that the functional modules important for expression from these promoters are concentrated upstream of the transcription start site; however the extent and composition of these modules in terms of the cis-acting elements they contain was different for each. For the UL37 promoter, both a HiNF-P factor binding site (-53 to -58 bp) and the TATA homology (-22 to -27) were required for any detectable expression, while an Sp1 binding site at -123 augmented this but was not absolutely required. In contrast, the only functional elements crucial for expression from the UL50 promoter were the TATA box (-25 to -31) and an Sp1 binding site at -117 bp relative to the cap site. Despite differences in detail, when the functional architecture of these two early promoters were compared to the extensively characterized HSV-1 thymidine kinase (UL23) promoter, class-specific similarities are clearly apparent.
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PMID:Functional modules important for activated expression of early genes of herpes simplex virus type 1 are clustered upstream of the TATA box. 965 2

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity. We have isolated cDNAs from the Trypanosomatidae Leishmania major and Trypanosoma cruzi capable of complementing the deficiency of exonuclease III and dUTPase in the Escherichia coli mutant BW286. This double mutant is non-viable at 37 degreesC due to an accumulation of non-repaired sites following excision of uracil from DNA. The genes were expressed as beta-galactosidase-AP endonuclease fusion proteins and as such are active in repair of AP sites in E. coli. The Trypanosoma and Leishmania sequences have unique N-termini containing sequences that correspond to probable nuclear transport signals, while the C-terminal domains exhibit pronounced similarity to exonuclease III. The L.major gene was overexpressed as a histidine-tagged protein and recombinant enzyme exhibited endonuclease activity on AP DNA in vitro. Furthermore, expression of the enzymes in AP endonuclease-deficient E.coli mutants conferred significant resistance to killing by methylmethane sulphonate and peroxides. This study constitutes one of the first descriptions of DNA repair enzymes in these pathogenic organisms where oxidative stress is an important mechanism of both drug-mediated and intracellular killing.
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PMID:Apurinic/apyrimidinic endonuclease genes from the trypanosomatidae leishmania major and Trypanosoma cruzi confer resistance to oxidizing agents in DNA repair-deficient Escherichia coli. 988 72