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)

Among 54 transcripts expressed in a temporal cascade during lytic infection with bovine herpesvirus 1, we have previously identified three major immediate-early (IE) RNAs, IER4.2 (4.2 kb), IER2.9 (2.9 kb), and IER1.7 (1.6 to 1.8 kb depending on the virus strain) transcribed from the HindIII C genome region (U. V. Wirth, K. Gunkel, M. Engels, and M. Schwyzer, J. Virol. 63:4882-4889, 1989). Northern (RNA) blot, S1 nuclease protection, and primer extension analysis used in the present study demonstrated that all three IE transcripts were spliced and originated from two divergent transcription units with start sites located in the inverted repeat. Transcription unit 1 encoded two alternative spliced transcripts, IER4.2 and IER2.9, with a common exon 1 located at 0.797 to 0.795 map units (m.u.) and an exon 2 for IER4.2 (0.792 to 0.762 m.u.) in the inverted repeat; exon 2 for IER2.9 (0.754 to 0.738 m.u.) was located in the unique long sequence and transcribed in antisense orientation to latency-related RNA. Transcription unit 2 (0.818 to 0.836 m.u.), further characterized by cDNA cloning, encoded the spliced IER1.7 with three exons in the inverted repeat. Additional minor IE transcripts were interpreted as unspliced precursors and splicing variants. With regard to the number and layout of IE genes, bovine herpesvirus 1 occupies an intermediate position between pseudorabies virus and equine herpesvirus 1 on the one hand and varicella-zoster virus and herpes simplex virus type 1 on the other.
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PMID:The three major immediate-early transcripts of bovine herpesvirus 1 arise from two divergent and spliced transcription units. 184 84

The cis-acting DNA sequences and trans-acting proteins that control the expression of the major immediate early (IE) gene of varicella-zoster virus (VZV) were investigated. The location of the IE mRNA 5' terminus was determined by primer extension and S1 nuclease analyses and the functional activities of DNA sequences upstream of this site were analysed by a transfection assay. The VZV IE promoter exhibited low activity in BHK and HeLa cells, but was transactivated by the herpes simplex virus type 1 (HSV-1) virion protein Vmw65. DNA sequences between positions -131 and +57 were responsible for promoter activity, whereas sequences between -410 and -131 mediated the response to Vmw65. Two short elements in the -410 to -131 region formed protein-DNA complexes with HeLa cell nuclear proteins and formed a ternary complex when Vmw65 was added. One of the elements, ATGTAAATGAAAT, possessed a strong similarity to the HSV-1 TAATGARAT. The VZV homologue of Vmw65, encoded by open reading frame (ORF) 10, failed to trans-activate expression from HSV-1 or VZV IE promoters and did not form a ternary complex with functional TAATGARAT elements and HeLa cell proteins. Therefore, stimulation of VZV IE transcription by Vmw65 can occur by a mechanism similar to that employed by HSV-1, but VZV ORF 10 does not function as a trans-activator of IE gene expression.
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PMID:Control of expression of the varicella-zoster virus major immediate early gene. 215 1

Efficient cleavage and polyadenylation of eukaryotic messenger RNAs require at least two signal elements: an AAUAAA or closely related sequence located 7-30 base pairs (bp) upstream of the site of processing, and a G/U- or U-rich sequence located 3' to the cleavage site. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT-rich region. We have shown that the first AATAAA and the GT-rich region are essential for efficient processing, both in vivo and in vitro, whereas the second AATAAA does not appear to play any role in the formation of tk mRNA 3' ends. The failure of a signal containing only the second AATAAA and the GT-rich element to signal cleavage and polyadenylation suggested that these two elements might be too close together to constitute a functional polyadenylation signal. The experiments described in this report were directed at determining the effects on mRNA 3' end formation of alterations in spacing between signal elements. Wild-type tk contains 19 bp between these two elements. Constructs were made in which an AATAAA and the GT-rich region were separated by various distances ranging from 7 to 43 bp. The quantity and location of 3' ends of the tk mRNA produced by these constructs in Cos-1 cells were measured by S1 nuclease protection analysis. Signal efficiency was gradually reduced as the separation between the two signal elements was increased; with a separation of 43 bp, the signal functioned at approximately one-eighth the efficiency of the parental construction. Bringing the two signals closer together resulted in decreased signal efficiency; with a separation of 7 or 9 bp, no tk mRNA polyadenylated within the normal region was produced. Altering the sequences between these two elements without changing the distance had small effects on processing efficiency.
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PMID:Spatial constraints on polyadenylation signal function. 216 Sep 55

Matrix attachment regions (MARs) are DNA elements that dissect the genome into topologically separated domains by binding to a chromosomal skeleton. This study explored the putative influence of the MAR located 5' of the chicken lysozyme gene on expression of heterologous genes in heterologous cell systems. Expression of a construct with the chloramphenicol acetyltransferase (CAT) indicator gene controlled by the herpes simplex virus thymidine kinase promoter (TC) and a construct in which the same transcriptional unit is flanked by chicken lysozyme 5' MARs (MTCM) was assayed after stable transfection into rat fibroblasts. Median CAT activity per copy number in MTCM transfectants was elevated approximately 10-fold relative to that in TC transfectants. Total variation in normalized CAT activity decreased from more than 100-fold among TC transfectants to nearly 6-fold among MTCM transfectants. The steady-state level of transcripts and the relative rate of transcription were increased in MTCM transfectants, as shown by S1 nuclease and run-on transcription assays, respectively. The chicken lysozyme 5' MAR thus can confer elevated, less position-dependent expression on a heterologous promoter in cells of a different species by increasing the density of transcribing RNA polymerase molecules. MAR-mediated transcriptional enhancement suggests that MARs are important for gene expression and not just for DNA packaging.
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PMID:The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. 232 53

We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 nuclease protection assays. In all constructs, all polyadenylation signals were used. Increasing the number of poly(A) signals 3' to the tk-coding region did not affect the total amount of polyadenylated RNA produced, even with the weakest signal. Increasing the distance between two signals caused an increase in the use of the 5' signal and a decrease in the use of the 3' signal. Changing the distance between the 5' cap and first signal did not affect signal use. Analyses of cytoplasmic mRNA stability, nuclear RNA distribution, and transcription in the polyadenylation signal region indicated that the distribution of tk RNAs ending at different poly(A) sites was the result of poly(A) signal choice, not other aspects of RNA metabolism. Four possible mechanisms of polyadenylation signal recognition are discussed.
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PMID:Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals. 246 66

The nucleotide sequence and deduced amino acid sequence of a vaccinia virus gene from the SalI F fragment are shown. The predicted polypeptide shares 42% amino acid identity over a 200 amino acid region with Saccharomyces cerevisiae thymidylate kinase (TmpK) and has low homology with herpes simplex virus deoxypyrimidine kinase. Northern blotting and S1 nuclease protection showed that the TmpK gene is transcribed early during infection and mapped the mRNA 5' end to immediately upstream of the second inframe ATG codon of the open reading frame (ORF). The encoded polypeptide is predicted to be 204 amino acids long (23.2 kD) and is almost colinear with yeast TmpK. Vaccinia virus possesses genes for TK and TmpK, separated by 57 kilobases of DNA, which are co-ordinately expressed and the encoded enzymes perform sequential steps in the same biochemical pathway.
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PMID:Vaccinia virus encodes a thymidylate kinase gene: sequence and transcriptional mapping. 255 11

RNA transfer (Northern) blot analysis was used to perform the physical characterization of the transcript expressed in murine sensory nerve ganglia latently infected with herpes simplex virus type 1. Most of this latency-associated transcript (LAT) was isolated in the poly(A)- fraction from ganglia. A smaller RNA species was also detected at less than 10% the abundance of the major one. LAT was not detected with probes from DNA outside the limits of the larger species. In situ hybridization data correlated well with Northern blot analysis; however, low levels of hybridization were seen with probes immediately outside the region of viral DNA giving positive Northern blot signals. S1 nuclease and primer extension mapping were used to locate the 5' end of the LAT 510 bases to the left of a KpnI site at 0.783 map units. The 3' end of the major latency-associated species was mapped to just within a 310-base-pair SmaI fragment located 660 to 970 base pairs to the right of the SalI site at 0.790 map units. These data were correlated with an analysis of the sequence of the DNA encoding this transcript and its possible function in the latent phase of infection.
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PMID:Physical characterization of the herpes simplex virus latency-associated transcript in neurons. 283 80

We have mapped the termini and determined the relative abundance and ribosome density of the major cytoplasmic transcript of the DNA polymerase (pol) gene of herpes simplex virus type 1. Nuclease protection and primer extension analyses located the 5' end of the major pol transcript at two closely spaced sites 51 and 57 nucleotides to the left of a BamHI site at map position 0.413. S1-sensitive sites corresponding to additional minor transcripts were found to map further upstream within a palindromic sequence that contains a viral replication origin. The major 3' end was found to map 90 nucleotides upstream of a KpnI site at map position 0.439. Quantitative S1 nuclease assays revealed that pol transcripts were nearly as abundant as transcripts encoded by the viral thymidine kinase gene. However, relatively few pol transcripts were found on large polysomes at 5.5 h after infection, when pol transcripts were most abundant. This was in marked contrast to the polyribosome distribution of transcripts from the thymidine kinase gene and the major DNA-binding protein gene. These results and sequence features of the pol transcript suggest that pol expression is regulated, in part, at the level of translation.
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PMID:Analysis of the transcript of the herpes simplex virus DNA polymerase gene provides evidence that polymerase expression is inefficient at the level of translation. 283 6

Most eucaryotic mRNAs are polyadenylated. In higher eucaryotes, the sequence AATAAA is located 7 to 30 base pairs (bp) upstream from the site of processing and polyadenylation and is a critical part of the signal for processing and polyadenylation. Efficient cleavage and polyadenylation also require sequences downstream of polyadenylation sites. The herpes simplex virus type 1 thymidine kinase (tk) gene contains two copies of the AATAAA hexanucleotide and a GT box (18 of 19 consecutive residues are G or T) previously shown to be required for efficient processing and polyadenylation of tk mRNA (C. N. Cole and T. P. Stacy, Mol. Cell. Biol., 5:2104-2113). To define further the sequence requirements for efficient polyadenylation, we prepared linker scanning, internal deletion, and small insertion mutations in the polyadenylation region of the tk gene. These mutations were analyzed by S1 nuclease protection analysis of cytoplasmic RNA isolated from transfected Cos-1 monkey kidney cells. When the proximal AATAAA was deleted, no tk mRNA polyadenylated in the normal region was detected, whereas replacement of the second AATAAA with an XbaI linker had no effect on polyadenylation. When various portions of the GT box were replaced with linker, the amount of tk mRNA produced was reduced to 23 to 82% of the normal amount, but polyadenylation in the normal region was never abolished. Thus, no single portion of the GT box was absolutely required. In some cases, extended transcripts, polyadenylated at a cryptic site within pBR322, were detected. A spacing of 6 bp between AATAAA and the GT box was too short for efficient processing and polyadenylation. A spacing of 30 bp appeared to work almost as efficiently as did the wild-type spacing of 18 bp. Taken together, these results indicate that efficient polyadenylation requires both AATAAA and downstream GT-rich sequences. In addition, processing and polyadenylation are affected both qualitatively and quantitatively by sequences at polyadenylation sites and at more distant locations.
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PMID:Fine-structure analysis of the processing and polyadenylation region of the herpes simplex virus type 1 thymidine kinase gene by using linker scanning, internal deletion, and insertion mutations. 287 21

We have used partially purified preparations of RNA polymerase II from mock-infected and herpes simplex virus type 1-infected HEp-2 cells to transcribe the herpes simplex virus type 1 strain F BamHI Z DNA fragment containing the promoter for immediate-early RNA-5 (alpha 47), a 1.8-kilobase, spliced RNA. In agreement with the in vivo transcriptional regulation of herpes simplex virus type 1 immediate-early genes, electrophoretic analyses of runoff and truncated transcripts from this template showed that RNA polymerase II from mock-infected cells initiates transcription more selectively than does that from the herpes virus-infected cells at the immediate-early RNA-5 promoter. S1 nuclease mapping of the 5' ends of in vivo- and in vitro-synthesized mRNA placed the initiation site ca. 110 base pairs upstream from the previously published site and also demonstrated the presence of a second, smaller intervening sequence between this new cap site and the previously characterized intervening sequence. S1 analyses also suggested that some splicing of the larger but not the smaller intervening sequence occurred in vitro.
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PMID:In vitro transcription of herpes simplex virus genes: identification of a new initiation site and second intervening sequence in the immediate-early RNA-5 gene. 298 42


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