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)

In the proviral DNA of mouse mammary tumor virus (MMTV), sequences up to approximately equal to 200 base-pairs from the RNA start site are required for stimulation of transcription by glucocorticoid hormones in cultured cells. A total of 26 mutant plasmids with clustered point mutations or small deletions in the hormone control region of the MMTV long terminal repeat were constructed, linked to the coding portion of the Herpes simplex virus thymidine kinase gene, and introduced by transfection into LTK- cells. Transcription from mutant DNA in the presence or absence of hormone was quantified by S1 nuclease protection assays. Our analysis revealed the presence of at least three control elements that affect the extent of transcription stimulation by glucocorticoid hormones: (1) a distal element, between -181 and -172 base-pairs from the RNA initiation site. Linker scanning mutants in this segment have a reduction of up to 20-fold in the hormone response with respect to wild type. (2) An element around position -120, defined by a mutation of 4 base-pairs between -121 and -117, which causes a fivefold reduction. (3) An element from approximately equal to -78 to -70, defined by a mutant with also a roughly fivefold lower stimulation. The first two are included in areas that have been shown by others to interact in vitro with hormone-receptor complexes; the last one overlaps the in vitro binding site of a nuclear protein factor. A mutant lacking all three elements (-193 to -70) is completely non-inducible by glucocorticoids. Together with earlier results obtained with 5' deletion mutants, the data show that the largest contribution to the stimulatory response is made by the distal element, which however does require the presence of both more-proximal ones for the response to be maximal. In the absence of the distal one, the two proximal elements together produce a residual stimulation in the order of 5 to 10% of wild type, while the -70 element alone is ineffective. In addition, we show that a functional TATA homology is required for maximum stimulation. It appears that transcriptional regulation of MMTV by glucocorticoid hormones is achieved by the concerted action of multiple sequence modules, not all of which correspond to receptor binding sites in vitro.
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PMID:Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis. 302 41

Earlier reports have localized mutations which affect the processing and transport of herpes simplex virus 1 glycoproteins to a region located between the genes specifying glycoprotein B and the major viral DNA-binding protein (beta 8). The nucleotide sequence of this region contains a single long open reading frame encoding a 780-amino-acid protein with a predicted molecular weight of 83,845. To confirm the existence of this protein, rabbit polyclonal antibody was made against a synthetic peptide made according to the predicted sequence of a hydrophilic domain near the carboxy terminal of the protein. This antibody reacted with an infected cell protein of an apparent molecular weight of 95,500. We designated this protein infected cell protein 18.5 (ICP18.5). S1 nuclease analysis suggested that the 5.6-kilobase mRNA encoding ICP18.5 is initiated predominantly from one site, but three weaker initiation sites also seemed to occur within a 74-base-pair stretch of DNA. This gene appears to be conserved in the Epstein-Barr virus (EBV) genome, inasmuch as 174 of the 780 amino acids of ICP18.5 align with corresponding amino acids predicted by the EBV open reading frame BALF3. The EBV gene is located adjacent to the gene specifying a homolog of the herpes simplex virus 1 glycoprotein B.
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PMID:Transcription initiation sites and nucleotide sequence of a herpes simplex virus 1 gene conserved in the Epstein-Barr virus genome and reported to affect the transport of viral glycoproteins. 302 64

The initiation site for transcription of the herpes simplex virus type 1 (HSV-1) gene encoding the major DNA-binding protein. ICP8, was mapped by nuclease S1 analysis of RNA-DNA hybrids. When RNA isolated from cells infected with HSV-1 was used, one major start site of ICP8 gene transcription was mapped at 89 base pairs to the right of the BstEII site at 0.409 map units. In cells transfected with a cloned ICP8 gene, the same major start site was detected either in the presence or absence of the immediate-early (alpha) genes encoding ICP4 or ICP0, which have been shown to stimulate ICP8 gene expression in transfected cells. Both ICP4 and ICP0 stimulated the accumulation of the ICP8 gene transcripts in the transient expression system, and their effects were synergistic. By comparison of the sequence of the putative promoter region of the ICP8 gene with the promoter of the HSV-1 TK gene, a significant similarity was detected between the three transcriptional regulatory signals of the TK gene and the upstream sequences of the ICP8 gene. Analysis of promoters of other delayed-early (beta) genes showed that they all contained regions of significant homology with the distal signals of the upstream sequences of the TK or ICP8 gene.
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PMID:Mapping of the transcriptional initiation site of the herpes simplex virus type 1 ICP8 gene in infected and transfected cells. 302 91

Herpes simplex viruses encode a structural protein which induces, in trans, expression of alpha genes, the first set of genes to be expressed after infection of permissive cells. This protein, designated as the alpha-trans-inducing factor (alpha-TIF), maps within the BamHI F fragment, and its gene has been sequenced. In the course of mapping the domain of the alpha-TIF gene, it was noted that the intact BamHI fragment was consistently more effective than the complete domain of the alpha-TIF gene in inducing expression of alpha genes. Cotransfections of DNA fragments containing an alpha indicator gene and the alpha-TIF gene with various regions of the BamHI F DNA fragment revealed that the sequences located 3' to the alpha-TIF gene raised the activity of the alpha-TIF gene to nearly the same level as that of the intact BamHI F fragment. The nucleotide sequence and S1 nuclease mapping analyses revealed the presence of two transcribed open reading frames capable of encoding polypeptides with translated molecular weights of 77,357 and 70,527. To determine whether the effect of these sequences in trans on alpha-TIF-mediated induction of alpha genes was due to expression of these genes or competition for transcriptional factors, we constructed plasmids that contained both genes. Into each or both of these genes we inserted, near the translation initiation sites, 14-base-pair linkers carrying translational stop codons (TAG) in all three reading frames. Analyses of these plasmids indicated that the gene encoding the 70,527-molecular-weight polypeptide reduced alpha-TIF-dependent induction of alpha genes, whereas the gene encoding the 77,357-molecular-weight polypeptide increased this activity. Insertion of the stop codons abolished these activities.
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PMID:Characterization and nucleotide sequence of two herpes simplex virus 1 genes whose products modulate alpha-trans-inducing factor-dependent activation of alpha genes. 302 33

This paper describes experiments to test the ability of a number of viruses of the Herpes group, and also Adenovirus-2 and SV40, to activate transcription from the Herpes simplex virus-1 glycoprotein D and the rabbit beta-globin promoters. Plasmids containing these genes were transfected into HeLa cells which were then infected with various viruses. Transcriptional activation in trans of the plasmid-borne promoters was monitored by quantitative S1 nuclease analysis of total cytoplasmic RNA isolated after infection. The results showed that Herpes simplex viruses 1 and 2, Pseudorabies virus, Variella Zoster virus, Human Cytomegalovirus, Equine herpes virus-1 and Adenovirus-2 activate transcription from both promoters tested. In contrast, SV40 did not activate transcription in trans in this assay. The possible mechanisms of this activation are discussed.
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PMID:Trans activation of plasmid-borne promoters by adenovirus and several herpes group viruses. 608 5

cAMP stimulates the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in rat liver. We have investigated the nucleotide sequences required for regulation of PEPCK gene expression by cAMP. A chimeric gene was constructed in which a 620-base pair fragment of the 5'-end of the PEPCK gene (including 547 base pairs of 5'-flanking sequence) was ligated to the herpes simplex virus thymidine kinase (TK) structural gene. The PEPCK promoter fragment was introduced either in the proper orientation for transcription of the TK gene or in the opposite orientation. These fusion genes and the parent vector, pOPF, which contains the intact TK gene, were transfected individually into TK-deficient FTO-2B rat hepatoma cells. FTO-2B cells contain an active endogenous PEPCK gene which is stimulated by cAMP. Cells were selected in HAT medium and grown either as mass cell cultures or as individual clones. Dibutyryl cyclic AMP (Bt2cAMP) plus theophylline (16 h) stimulated TK activity 1.6-6.1-fold in cell lines transfected with the PEPCK-TK fusion gene containing the PEPCK promoter fragment in the correct orientation. However, the intact TK gene was not induced by Bt2cAMP after transfection, nor was there any expression of the PEPCK-TK fusion gene in cells which contained the PEPCK promoter fragment in the wrong transcriptional orientation. Bt2cAMP also increased the levels of TK mRNA in cells transfected with the PEPCK-TK fusion gene, but not in cells transfected with the intact TK gene. The chimeric PEPCK-TK mRNA initiated at the PEPCK start site, as determined by S1 nuclease mapping. There was no relationship between the number of copies of the PEPCK-TK gene integrated in the various cell lines and either the basal level of TK activity or its inducibility of Bt2cAMP.
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PMID:Identification of a cAMP regulatory region in the gene for rat cytosolic phosphoenolpyruvate carboxykinase (GTP). Use of chimeric genes transfected into hepatoma cells. 609 Apr 58

The Chinese hamster ovary adenine phosphoribosyl transferase gene (aprt) was reengineered to be flanked by sequences from the thymidine kinase (tk) gene of herpes simplex virus. This construct was cotransfected with DNA from herpes simplex virus type 1, and after 3 days, virus was harvested and Tk- plaques were selected after the virus was plated on Tk- cells in the presence of bromodeoxycytosine. Recombinant viruses were identified by dot-blot hybridization, and the arrangement of aprt and tk sequences were determined by Southern blot hybridization. Analysis of the recombinants revealed that acquisition of aprt sequences resulted from insertional inactivation of the tk locus as a consequence of homology-based recombination. Recombination was precise, as evidenced by the failure to detect plasmid sequences or the synthetic restriction endonuclease sites that bounded the mutant tk gene in the aprt-tk construct. Infection of Aprt- mouse or Chinese hamster ovary cells with UV-irradiated virus and selection in medium containing azaserine and adenine resulted in the survival of numerous colonies that stably express the aprt gene. Transformed cells synthesized an aprt mRNA that is identical to wild-type mRNA as determined by Northern blot and S1 nuclease analyses. Cells lytically infected with the recombinant virus do not appear to transcribe the aprt gene. Thus, infected cells differentiate between virus and foreign promoters even when a cellular gene is cis to the virus chromosome.
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PMID:Transduction of the Chinese hamster ovary aprt gene by herpes simplex virus. 609 82

Cloned complementary DNAs encoding chicken ovalbumin, chicken prelysozyme and calf preprochymosin, prochymosin and chymosin were inserted downstream from various viral promoters in modified recombinant "shuttle" vectors. Microinjection of the ovalbumin, prelysozyme and preprochymosin constructs into the nuclei of Xenopus laevis oocytes resulted in the synthesis, segregation in membranes and secretion into the extracellular medium of ovalbumin, lysozyme and prochymosin, respectively. Judging from molecular weight estimations, lysozyme and prochymosin were correctly proteolytically processed while ovalbumin, which lacks a cleavable signal sequence, was glycosylated. Injection of the DNA construct encoding prochymosin without its signal sequence resulted in synthesis of prochymosin protein that was localized exclusively in the oocyte cytoplasm. No immunospecific protein was detected after injection of the DNA encoding mature chymosin. In terms of protein expression in oocytes, the Herpes simplex thymidine kinase (TK) promoter was up to sevenfold more effective than the simian virus 40 (SV40) early promoter, and equally as effective as the Moloney murine sarcoma virus long terminal repeat element. Where tested, protein expression in oocytes was much reduced if DNA sequences encoding the SV40 small t intron and its flanking sequences were present in the constructs. S1 nuclease mapping of transcripts produced after injection of DNAs containing the TK promoter indicated that the majority of transcripts initiated at, or within, two bases of the known "cap" site. However, minor transcripts initiating upstream from this site were observed and one (or more) of these transcripts was responsible for the synthesis of an ovalbumin polypeptide containing a 51 amino acid N-terminal extension. This extended protein remained in the oocyte cytosol. When ovalbumin cDNA was inserted into the vectors with opposite polarity to the viral promoter, expression in oocytes resulted in the predominant synthesis and secretion of a variant ovalbumin with a 21 amino acid N-terminal extension, although some full-length ovalbumin was also synthesized and secreted. S1 mapping revealed the presence, in these oocytes, of transcripts of predicted polarity initiating 118 bases upstream from the wild type ovalbumin initiator ATG, at a previously unreported SV40 "promoter". No protein synthesis was detected after the injection of these reverse-orientation constructs into baby hamster kidney (BHK-21) cells.
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PMID:Efficient expression of cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes. 609 86

The BglII N DNA fragment (0.580 to 0.620 map units) of herpes simplex virus type 2 strain (333) is of interest because of its transforming potential. This fragment contains either partial or the complete coding sequences for nine mRNA transcripts that can be detected during a lytic infection. Subclones of the BglII N DNA fragment were generated in plasmid vectors, and the approximate locations of the mRNA transcripts were mapped by RNA blot hybridization technology. Precise 5' or 3' ends (or both) of these mRNA species were determined by S1 nuclease mapping, using the BglII N subclones as DNA probes. At least four mRNA transcripts are fully encoded in the BglII N fragment. The coding regions for all of the mRNA transcripts are densely packaged along the BglII N fragment with less than 150 base pairs between neighboring mRNA ends. Analysis of both neutral and alkaline gels failed to reveal the presence of any detectable introns. This manuscript reports a detailed transcription map for this region.
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PMID:Characterization of mRNAs that map in the BglII N fragment of the herpes simplex virus type 2 genome. 620 12

This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The position on the DNA sequence corresponding to the 5' and 3' termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the 5' terminus of tk mRNA to a UGA stop codon approximately 70 nucleotides from the poly-A addition site. The results of these experiments indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.
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PMID:The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene. 625 56


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