Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Regulation of intracellular cholesterol metabolism has been studied in Epstein-Barr virus-transformed lymphoblasts from patients with Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD) disease. Addition of LDL to normal lymphoblasts cultured in lipoprotein-deficient medium increased cholesterol esterification 10-fold (to a maximum of 1.0 nmol/h/mg protein at 15 h), while little stimulation was seen in NPC cells. The response by NPD lymphoblasts was intermediate, reaching approximately half of normal values by 14-24 h. Lymphoblasts from both NPC and NPD obligate heterozygotes exhibited 50% of normal LDL-stimulated cholesterol esterification at 6 h, when activity was < 10% of normal values in patient cells. Fluorescence staining with filipin indicated excessive intracellular accumulation of LDL-derived cholesterol in both NPC and NPD lymphoblasts. Downregulation of LDL receptor mRNA levels by LDL, measured by S1 nuclease protection assay, was also impaired in NP lymphoblasts and fibroblasts (NPC > NPD), although a similar rate of receptor protein down-regulation by LDL (t1/2 = 10-15 h) was observed in normal and NP lymphoblasts. In contrast, LDL down-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not appear to be affected in NP cells: LDL produced a 3-fold (lymphoblasts) or > 10-fold (fibroblasts) decrease by 12 h in both normal and affected cells. Thus, NPC and NPD lymphoblasts exhibit distinct defects in cholesterol esterification and storage, similar to those observed in mutant fibroblasts. Other regulatory responses are also impaired in NPC lymphoblasts but appear to be less affected in NPD cells. Lymphoblasts should provide a valuable immortalized cell line model for study of defective regulation of cholesterol esterification and transport in Niemann-Pick type II disease, and may also be suitable for diagnosis and carrier detection.
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PMID:Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann-Pick type C and type D patients. 820 65

Pulsed-field agarose gel electrophoresis showed that fragmentation of chromosomal DNA in Raji cells was induced by infection with the P3HR-1 strain of Epstein-Barr virus (EBV). S1 nuclease treatment of the agarose plugs containing cells suggested that the majority of DNA fragments did not contain single-strand gaps. Chromosomal DNA fragmentation was inhibited by cycloheximide, indicating that protein synthesis was required for DNA fragmentation. Phosphonoacetic acid, an inhibitor of EBV DNA polymerase, did not inhibit fragmentation of chromosomal DNA. These findings suggest that EBV-specific early proteins participate in fragmentation of chromosomal DNA. Chromosomal DNA of P3HR-1 cells was also fragmented by treatment with n-butyrate plus 12-O-tetradecanoylphorbol-13-acetate (TPA), which induced activation of latent EBV genome following viral replication. In addition, fragmentation of DNA preceded cell death during lytic infection. These results suggest that fragmentation of chromosomal DNA is generally induced during EBV replication and probably contributes to the cytopathic effect of EBV. The role of DNA fragmentation in death of infected cells is discussed in relation to apoptosis.
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PMID:Epstein-Barr virus induces fragmentation of chromosomal DNA during lytic infection. 823 Apr 85

Immediate-early (IE) RNA 2, the less abundant of two bovine herpesvirus 4 (BHV-4) RNAs detected in Madin-Darby bovine kidney cells infected in the presence of cycloheximide, is a 1.8-kb cytoplasmic polyadenylated RNA transcribed from the 8.3-kb HindIII fragment F. The structure of IE RNA 2 has been determined by S1 nuclease and exonuclease VII mapping, primer extension analysis, and sequencing of a partial cDNA. IE RNA 2 consists of a short, approximately 60-nucleotide 5' exon spliced to a 1.8-kb 3' exon. DNA sequence analysis revealed an open reading frame encoding 551 amino acids with sequence homology to the Epstein-Barr virus (EBV) R transactivator and its homolog in herpesvirus saimiri, HVS.R.IE 2 and HVS.R show higher homology to each other than to the EBV R transactivator. The homology is highest in the approximately 320 amino-terminal amino acids. All three proteins have acidic carboxyl termini but have little amino acid sequence homology in this region. In transient expression cotransfection assays, IE 2 activated expression from the BHV-4 early promoter-regulatory region of the major DNA-binding protein homolog over 100-fold in bovine turbinate cells. IE 1 was not necessary for this transactivation and did not augment it. However, IE 2 did not transactivate EBV or herpesvirus saimiri early promoter-regulatory regions that are transactivated by the EBV R transactivator or HVS.R.
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PMID:Characterization of a bovine herpesvirus 4 immediate-early RNA encoding a homolog of the Epstein-Barr virus R transactivator. 838 Apr 65

During Epstein-Barr virus (EBV) latent infection of B lymphocytes in vitro, six viral nuclear antigens (EBNAs) are expressed from one of two promoters, Cp or Wp, whose activities have previously been shown to be mutually exclusive in established lymphoblastoid cell lines. Initially after infection, the EBNA genes are transcribed from Wp, which is present in multiples copies within the major internal repeat of EBV. Approximately 48 to 72 h postinfection, Wp is downregulated, with a corresponding increase in transcription from Cp. An EBNA2-responsive enhancer exists upstream of Cp, and a role for EBNA2 in the induction of Cp activity during the establishment of viral latency has previously been proposed (Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 87:1725-1729, 1991). To critically assess the potential role for this enhancer region in determining relative usage of Cp and Wp, an EBNA2 enhancer deletion mutant virus was generated. Lymphoblastoid cell lines were screened by PCR and Southern blotting for the presence of mutant virus harboring the EBNA2 enhancer deletion. A quantitative S1 nuclease protection assay was developed to allow comparison of relative Cp and Wp activities for the cell lines containing mutant virus and those of the wild-type recombinants which lacked the enhancer deletion. In general, the wild-type recombinants had higher levels of Cp-initiated transcripts than Wp-initiated transcripts. In contrast, the Cp EBNA2 enhancer deletion mutants exhibited a strong bias toward Wp activity. Notably, only the first Wp (oriP-proximal Wp; Wp1) appears active in these mutants. S1 nuclease protection assays using a probe which hybridizes to the W2 exon, contained in both Cp- and Wp-initiated transcripts, indicated that the total level of transcription from Cp and Wp remained the same in wild-type and EBNA2 enhancer mutant cell lines. The presence of both Cp and Wp activity in the wild-type recombinants, as well as in newly derived lymphoblastoid cell lines established with the prototype B95.8 virus, demonstrated that Cp and Wp activities are not always mutually exclusive.
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PMID:B-cell lines immortalized with an Epstein-Barr virus mutant lacking the Cp EBNA2 enhancer are biased toward utilization of the oriP-proximal EBNA gene promoter Wp1. 937 70

The Epstein-Barr virus (EBV) EBNA2 protein is a transcriptional activator that controls viral latent gene expression and is essential for EBV-driven B-cell immortalization. EBNA2 is expressed from the viral C promoter (Cp) and regulates its own expression by activating Cp through interaction with the cellular DNA binding protein CBF1. Through regulation of Cp and EBNA2 expression, EBV controls the pattern of latent protein expression and the type of latency established. To gain further insight into the important regulatory elements that modulate Cp usage, we isolated and sequenced the Cp regions corresponding to nucleotides 10251 to 11479 of the EBV genome (-1079 to +144 relative to the transcription initiation site) from the EBV-like lymphocryptoviruses found in baboons (herpesvirus papio; HVP) and Rhesus macaques (RhEBV). Sequence comparison of the approximately 1,230-bp Cp regions from these primate viruses revealed that EBV and HVP Cp sequences are 64% conserved, EBV and RhEBV Cp sequences are 66% conserved, and HVP and RhEBV Cp sequences are 65% conserved relative to each other. Approximately 50% of the residues are conserved among all three sequences, yet all three viruses have retained response elements for glucocorticoids, two positionally conserved CCAAT boxes, and positionally conserved TATA boxes. The putative EBNA2 100-bp enhancers within these promoters contain 54 conserved residues, and the binding sites for CBF1 and CBF2 are well conserved. Cp usage in the HVP- and RhEBV-transformed cell lines was detected by S1 nuclease protection analysis. Transient-transfection analysis showed that promoters of both HVP and RhEBV are responsive to EBNA2 and that they bind CBF1 and CBF2 in gel mobility shift assays. These results suggest that similar mechanisms for regulation of latent gene expression are conserved among the EBV-related lymphocryptoviruses found in nonhuman primates.
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PMID:Transcriptional activation signals found in the Epstein-Barr virus (EBV) latency C promoter are conserved in the latency C promoter sequences from baboon and Rhesus monkey EBV-like lymphocryptoviruses (cercopithicine herpesviruses 12 and 15). 984 97

We determined the structure of a 1.1-kb cytoplasmic polyadenylated RNA transcribed from a region of the bovine herpesvirus 4 (BHV-4) genome not conserved among gammaherpesviruses by sequencing its cDNA, by S1 nuclease analysis, and by primer extension analysis. We found that the RNA consists of a short, approximately 193-nucleotide (nt), 5' exon spliced to a 799-nt 3' exon and contains two short (53 and 57 codons) overlapping open reading frames (ORFs). The 53-codon ORF was previously designated BORFE1. Neither ORF exhibits detectable amino acid sequence homology with ORFs of other gammaherpesviruses. The 1.1-kb RNA promoter-regulatory region was specifically transactivated by the BHV-4 IE2 gene product, a homolog of the Epstein-Barr virus R transactivator, in cotransfection assays. In gel retardation experiments, IE2 protein formed a complex with DNA in a 129-bp fragment between -23 and -151 relative to the transcription start site of the 1.1-kb RNA, and less efficiently with a 57-bp subfragment between -78 and -22. A sequence similar to sequences of IE2-binding fragments of other BHV-4 IE2 responsive promoters was found partly in the 57-bp subfragment, extending into the portion of the 129-bp fragment not found in the 57-bp fragment. The 129-bp fragment, but not the 57-bp fragment, was sufficient for transactivation of a promoterless chloramphenicol acetyl transferase (CAT) reporter gene by IE2. However, the 129-bp fragment did not function efficiently as an IE2-responsive enhancer when inserted approximately 140 base pairs (bp) 5' to the transcription start site of a CAT reporter gene driven by an enhancerless simian virus 40 early promoter. Based on this and other observations, we propose that IE2 functions as a promoter factor rather than an enhancer factor.
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PMID:Characterization of a bovine herpesvirus 4(BHV-4) 1.1-kb RNA and its transactivation by BHV-4 immediate-early 2 gene product. 993 Jan 95

Gene 50 is the only immediate-early gene that appears to be conserved among the characterized gammaherpesviruses. It has recently been demonstrated for the human viruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) that ectopic expression of the gene 50-encoded product in some latently infected cell lines can lead to the induction of virus replication, indicating that gene 50 is likely to play a pivotal role in regulating gammaherpesvirus reactivation. Here we demonstrate that the murine gammaherpesvirus 68 (gammaHV68) gene 50 is an immediate-early gene and that transcription of gammaHV68 gene 50 leads to the production of both spliced and unspliced forms of the gene 50 transcript. Splicing of the transcript near the 5' end serves to extend the gene 50 open reading frame, as has been observed for the gene 50 transcripts encoded by KSHV and herpesvirus saimiri (Whitehouse et al., J. Virol. 71:2550-2554, 1997; Lukac et al., Virology 252:304-312, 1998; Sun et al., Proc. Natl. Acad. Sci. USA 95:10866-10871, 1998). Reverse transcription-PCR analyses, coupled with S1 nuclease protection assays, provided evidence that gene 50 transcripts initiate at several sites within the region from bp 66468 to 66502 in the gammaHV68 genome. Functional characterization of the region upstream of the putative gene 50 transcription initiation site demonstrated orientation-dependent promoter activity and identified a 110-bp region (bp 66442 to 66552) encoding the putative gene 50 promoter. Finally, we demonstrate that the gammaHV68 gene 50 can transactivate the gammaHV68 gene 57 promoter, a known early gene target of the gene 50-encoded transactivator in other gammaherpesviruses. These studies show that the gammaHV68 gene 50 shares several important molecular similarities with the gene 50 homologs in other gammaherpesviruses and thus provides an impetus for future studies analyzing the role of the gammaHV68 gene 50-encoded protein in acute virus replication and reactivation from latency in vivo.
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PMID:Characterization of gammaherpesvirus 68 gene 50 transcription. 1064 77


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