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:3.1.30.1 (S1 nuclease)
3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene (pol) encoding the Epstein-Barr virus (EBV) DNA polymerase is a member of the "early" class of viral genes which are expressed shortly after activation of latent virus infection. First, mRNA from the EBV-producing cell line, B95-8, treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate to induce lytic replication and expression of this gene was analyzed. Northern (RNA) analysis revealed a message of 3.7 kb found only in induced cells. 5' mapping of pol mRNA by S1 nuclease and primer extension analyses indicates that transcription initiates at tightly clustered sites within a G + C-rich region 126 bp upstream of the open reading frame. The same initiation region was identified in two other EBV-infected cell lines, P3HR1 and Raji, after induction. Second, a 1.29-kb genomic fragment containing this region, when cloned upstream of the chloramphenicol acetyltransferase reporter gene, demonstrated promoter activity in lymphoid cells cotransfected with pEBV-RZ, a genomic expression construct that includes genes for the EBV immediate-early transactivator proteins, BZLF-1 and BRLF-1. Within the upstream 1.29-kb sequence, two regions of 140 bp and 101 bp appear to be needed for promoter activity. These results demonstrate that unlike most EBV genes studied thus far, the pol gene contains multiple transcriptional start sites. The upstream regulatory region of the promoter for the pol gene does not contain canonical promoter elements such as TATA and CAAT boxes and, furthermore, is not constitutively active but requires transactivation by two or more viral proteins.
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PMID:Regulation of the Epstein-Barr virus DNA polymerase gene. 131 4

In Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cell lines exhibiting the latency I form of infection (i.e., EBV nuclear antigen 1 [EBNA1] positive in the absence of other latent proteins), the EBNA1 mRNA has a unique BamHI Q/U/K splice structure and is expressed from a novel promoter, Fp, located near the BamHI FQ boundary. This contrasts with the situation in EBV-transformed lymphoblastoid cell lines (LCLs) exhibiting the latency III form of infection (i.e., positive for all latent proteins), in which transcription from the upstream Cp or Wp promoters is the principal source of EBNA mRNAs. We carried out cDNA amplifications with oligonucleotide primer-probe combinations to determine whether Fp is ever active in an LCL environment. The results clearly showed that some LCLs express a Q/U/K-spliced EBNA1 mRNA in addition to the expected Cp/Wp-initiated transcripts; this seemed inconsistent with the concept of Cp/Wp and Fp as mutually exclusive promoters. Here we show that Fp is indeed silent in latency III cells but is activated at an early stage following the switch from latency III into the virus lytic cycle. Four pieces of evidence support this conclusion: (i) examples of coincident Cp/Wp and Fp usage in LCLs are restricted to those lines in which a small subpopulation of cells have spontaneously entered the lytic cycle; (ii) transcripts initiating from Fp can readily be demonstrated in spontaneously productive lines by S1 nuclease protection; (iii) the presence of Fp-initiated transcripts is not affected by acyclovir blockade of the late lytic cycle; and (iv) infection of latently infected LCLs with a recombinant vaccinia virus encoding the EBV immediate-early protein BZLF1, a transcriptional transactivator which normally initiates the lytic cycle, results in the appearance of the diagnostic Q/U/K-spliced transcripts.
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PMID:The Epstein-Barr virus (EBV) nuclear antigen 1 BamHI F promoter is activated on entry of EBV-transformed B cells into the lytic cycle. 133 31

Mu transcription occurs in three phases: early, middle, and late. Middle transcription occurs in the region of the C gene, which encodes the transactivator for late transcription. A middle promoter, Pm, was previously localized between 0.28 and 1.2 kilobase pairs upstream of C. We used S1 nuclease mapping with both unlabeled and radiolabeled capped RNAs from induced lysogens to characterize C transcription and identify its promoter. The C transcription initiation site was localized to a 4-base-pair region, approximately 740 base pairs upstream of C within the region containing Pm. Transcription of C was activated between 4 and 8 min after induction of cts and Cam lysogens and increased throughout the lytic cycle. Significant C transcription did not occur in replication-defective Aam lysogens. These kinetic and regulatory characteristics identify the C transcript as a middle RNA species and demonstrate that Pm is the C promoter. DNA sequence analysis of the Pm region showed a good -10, but poor -35, site homology to the Escherichia coli RNA polymerase consensus sequence. In addition, the sequence demonstrated that C is the distal gene in a middle operon containing several open reading frames. S1 mapping also showed an upstream transcript with a 3' end in the Pm region at a sequence strongly resembling a Rho-independent terminator. The regulatory characteristics of this RNA are consistent with this terminator, t9.2, being the early operon terminator.
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PMID:Characterization of the C operon transcript of bacteriophage Mu. 213 35

Cells latently infected with Epstein-Barr virus (EBV) can be activated to express lytic-cycle polypeptides by the introduction of the EBV-encoded Z transactivator, indicating that this protein has a pivotal role in virus reactivation. We examined the target specificity of the Z transactivator in short-term contransfection assays and found that the most responsive target to Z transactivation was the divergent NotI repeat promoter, located within the EBV BamHI H fragment. In contrast, target plasmids containing the cat gene linked to heterologous viral promoters were not activated by cotransfection with the Z gene. S1 nuclease analysis of RNA from chemically induced B95-8 cells and from Vero cells cotransfected with NotI repeat promoter-CAT and Z showed that Z transactivation increased the level of correctly initiated, stable RNA transcripts. The NotI repeat gene (ntr) gives rise to a highly abundant mRNA species after chemical induction of lytic virus replication, but no protein product had been previously identified. Using monospecific antiserum raised against a synthetic peptide from the BHLF1 open reading frame, we demonstrated that the ntr gene encodes a protein product that is found in nuclear patches colocalizing with nucleoli. A series of deletions introduced into the upstream sequences of the NotI-repeat-promoter revealed two separate Z-response regions. The minimal promoter region between -7 and -155 of the leftward RNA cap site and an upstream region between -644 and -902 were both independently capable of conferring Z responsiveness. However, the minimal region, which was activated by Z cotransfection in Vero cells, was poorly responsive in lymphocytes, whereas the response of the far-upstream region to Z cotransfection was lymphocyte specific. In its human host, EBV infects both epithelial and lymphocyte populations. This dual lifestyle may have led to the evolution of multiple Z-response signals that enable the Z transactivator to interact with both cell-specific promoter and enhancer factors.
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PMID:Responsiveness of the Epstein-Barr virus NotI repeat promoter to the Z transactivator is mediated in a cell-type-specific manner by two independent signal regions. 254 12

A transient DNA replication assay was used to identify genes located within m.u. 90.5-7.0 of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that influenced replication of a reporter plasmid containing an OpMNPV origin of replication, when cotransfected into uninfected Lymantria dispar cells. The viral transactivator ie-1 and a 2.4-kb subclone were found to be essential for replication. The 2.4-kb region was sequenced and open reading frames were identified. Replication assays using subclones from this region identified a gene called late expression factor 2 (lef-2), as the essential replication gene. The OpMNPV lef-2 gene encodes a protein with a predicted molecular weight of 22.7 kDa (204 amino acids) and exhibits 54.7% amino acid sequence identity with its homolog in the genome of the Autographa californica MNPV. Transcriptional mapping using both Northern blot and S1 nuclease protection assays demonstrated that OpMNPV lef-2 was expressed at both early and late times postinfection as a transcript of about 1.6 kb. The early transcript initiated approximately 30 nt downstream of a TAATA box, whereas the late transcript initiated from within a late promoter consensus motif. In addition, we identified three genes stimulatory for DNA replication including two OpMNPV transcriptional activators (ie-2 and p34) and Op-iap, which is the functional analog of the AcMNPV p35 gene that inhibits apoptosis in AcMNPV-infected Spodoptera frugiperda cells.
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PMID:Replication of Orgyia pseudotsugata baculovirus DNA: lef-2 and ie-1 are essential and ie-2, p34, and Op-iap are stimulatory genes. 757 35

Four of the 68 varicella-zoster virus (VZV) unique open reading frames (ORFs), i.e., ORFs 4, 61, 62, and 63, encode proteins that influence viral transcription and are considered to be positional homologs of herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins. In order to identify the elements that regulate transcription of VZV ORFs 4 and 63, the encoded mRNAs were mapped in detail. For ORF 4, a major 1.8-kb and a minor 3.0-kb polyadenylated [poly(A)+] RNA were identified, whereas ORF 63-specific probes recognized 1.3- and 1.9-kb poly(A)+ RNAs. Probes specific for sequences adjacent to the ORFs and mapping of the RNA 3' ends indicated that the ORF 4 RNAs were 3' coterminal, whereas the RNAs for ORF 63 represented two different termination sites. S1 nuclease mapping and primer extension analyses indicated a single transcription initiation site for ORF 4 at 38 bp upstream of the ORF start codon. For ORF 63, multiple transcriptional start sites at 87 to 95, 151 to 153, and (tentatively) 238 to 243 bp upstream of the ORF start codon were identified. TATA box motifs at good positional locations were found upstream of all mapped transcription initiation sites. However, no sequences resembling the TAATGARAT motif, which confers IE regulation upon HSV-1 IE genes, were found. The finding of the absence of this motif was supported through analyses of the regulatory sequences of ORFs 4 and 63 in transient transfection assays alongside those of ORFs 61 and 62. Sequences representing the promoters for ORFs 4, 61, and 63 were all stimulated by VZV infection but failed to be stimulated by coexpression with the HSV-1 transactivator Vmw65. In contrast, the promoter for ORF 62, which contains TAATGARAT motifs, was activated by VZV infection and coexpression with Vmw65. These results extend the transcriptional knowledge for VZV and suggest that ORFs 4 and 63 contain regulatory signals different from those of the ORF 62 and HSV-1 IE genes.
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PMID:Transcriptional mapping of the varicella-zoster virus regulatory genes encoding open reading frames 4 and 63. 818 96

The major immediate-early (MIE) transactivator proteins of cytomegaloviruses (CMV) play a pivotal role in the initiation of virus-host cell interactions. Therefore, cis- and trans-acting factors influencing the expression of these proteins through their upstream promoter-enhancer regions are important determinants of the outcome of virus infection. S1 nuclease analysis and in vitro transcription assays with the MIE (or IE94) transcription unit of simian CMV (SCMV) (Colburn) revealed a single prominent mRNA start site associated with a canonical TATATAA motif. This initiator region lies adjacent to a 2,400-bp 5'-upstream noncoding sequence that encompasses a newly identified 1,000-bp (A+T)-rich segment containing intrinsically bent DNA (domain C), together with the previously described proximal cyclic AMP response element locus (domain A) and a tandemly repeated nuclear factor I binding site cluster (domain B). Deleted MIE reporter gene constructions containing domain A sequences only yield up to 4-fold stronger basal expression in Vero cells than the intact simian virus 40 promoter-enhancer region, and sequences from position -405 to -69 (ENH-A1) added to a minimal heterologous promoter produced a 50-fold increase of basal expression in an enhancer assay. In contrast, neither the nuclear factor I cluster nor the bent DNA region possessed basal enhancer properties and neither significantly modulated the basal activity of the ENH-A1 segment. A second segment of domain A from position -580 to -450 was also found to possess basal enhancer activity in various cell types. This ENH-A2 region contains three copies of a repeated element that includes the 10-bp palindromic sequence CCATATATGG, which resembles the core motif of serum response elements and proved to bind specifically to the cellular nuclear protein serum response transcription factor. Reporter gene constructions containing four tandem copies of these elements displayed up to 13-fold increased basal enhancer activity and 18-fold tetradecanoyl phorbol acetate responsiveness in U937 cells, but an ENH-A2 DNA segment encompassing two of the core serum response transcription factor binding sites failed to respond to serum induction in NIH 3T3 cells. Although there are overall similarities in the organizations of both the MIE enhancers and MIE transcription units among human CMV, SCMV, and murine CMV, the specific arrangements of repetitive motifs are quite different, and the bent DNA and ENH-A2 domains appear to be unique to SCMV.
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PMID:Identification of a large bent DNA domain and binding sites for serum response factor adjacent to the NFI repeat cluster and enhancer region in the major IE94 promoter from simian cytomegalovirus. 838 90

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

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is highly conserved within vertebrate species. Its pattern of expression in vivo seems to be tightly regulated both developmentally and in a tissue-specific manner, but shows differences with species. To identify transcriptional regulatory elements in the CFTR promoter region, we have used a combined approach based both on the analysis of the chromatin structure in vivo in rat tissues and on evolutionary clues (i.e. phylogenetic footprinting). In CFTR-expressing tissues, 15 DNase I-hypersensitive sites were identified within a 36 kb region encompassing exon 1. Eleven of them are clustered in a 3.5 kb region that exhibits eleven phylogenetic footprints observed when comparing sequences from eight mammalian species representing four orders (Primates, Artiodactylia, Lagomorpha and Rodentia). Comparison of the two sets of data allows the identification of two types of regulatory elements. Some are conserved between species, such as a non-consensus cAMP response element (CRE) and a PMA-responsive element (TRE) located respectively at positions -0.1 and -1.3 kb relative to ATG. Some are species-specific elements such as a 300 bp purine.pyrimidine (Pu.Py) stretch that is present only in rodents. Analysis of protein/DNA interactions in vitro with rat tissue protein extracts on the conserved elements revealed that the TRE site binds a specific heterodimeric complex composed of Fra-2, Jun D and a protein immunologically related to Jun/CRE-binding protein in the duodenum, whereas the CRE-like site binds ATF-1 ubiquitously. Functional analysis in Caco-2 cells showed that the CRE-like site supports a high basal transcriptional activity but is not able by itself to induce a response to cAMP, whereas the TRE site acts as a weak transactivator stimulated by PMA. Lastly, we found that the rodent-specific Pu.Py stretch confers nuclease S1 hypersensitivity under conditions of acidic pH and supercoiling. This indicates a non-B DNA conformation and thus reinforces the biological significance of non-random Pu.Py strand asymmetry in the regulation of transcription. Thus the tight transcriptional regulation of CFTR expression involves the combination of multiple regulatory elements that act in the chromatin environment in vivo. Some of them are conserved throughout evolution, such as the CRE-like element, which is clearly involved in the basal level of transcription; others are species-specific.
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PMID:Cross-species characterization of the promoter region of the cystic fibrosis transmembrane conductance regulator gene reveals multiple levels of regulation. 958 39

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


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