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

---

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

Studies with molecular and immunological techniques identified and mapped the transcript encoding glycoprotein D (gD) of equine herpesvirus 1 KyA, as well as two continuous gD antigenic determinants. Three mRNA species of 5.5, 3.8, and 1.7 kb overlap the gD open reading frame and are transcribed from the DNA strand encoding gD. Northern (RNA) blot hybridization with both DNA clones and riboprobes, as well as S1 nuclease analyses, showed the 3.8-kb mRNA to encode gD and to be synthesized as a late (beta-gamma) transcript. The 3.8-kb gD mRNA initiates within the US segment 91 and 34 nucleotides downstream of the CCAAT and TATA elements, respectively, and encodes a potential polypeptide of 392 amino acids. The termination site of this transcript maps within the terminal repeat at a site also used by the 5.5-kb mRNA and the IR6-encoded 1.2-kb mRNA, such that these three transcripts form a 3'-coterminal nested set. The extended size (2,250 nucleotides) of the 3' untranslated region of the gD transcript and its termination within the terminal repeat may result from the deletion of 3,859 bp, which eliminates two consensus polyadenylation signals downstream of the gD open reading frame of EHV-1 KyA. Use of antisera to synthetic peptides of 19 amino acids (residues 4 to 22) and 20 amino acids (residues 267 to 285) in Western immunoblot analyses revealed that gD is present in EHV-1 virions as a 55-kDa polypeptide. In addition, these antisera detected the 55-kDa protein as well as 58- and 47-kDa polypeptides in infected-cell extracts at late times of infection. Residues 4 to 22 make up a continuous neutralizing epitope of gD, since incubation of equine herpesvirus 1 with the anti-19-mer serum prior to infection results in reduced numbers of plaques and reduced levels of virus-encoded thymidine kinase. Complement is not required for neutralization mediated by the anti-19-mer serum.
...
PMID:Equine herpesvirus 1 glycoprotein D: mapping of the transcript and a neutralization epitope. 138 65

The 4.4-kb BamHI-E fragment of the orf virus (OV) genome contains three discrete open reading frames designated ORF-pp, ORF-1, and ORF-3, all of which are flanked by vaccinia virus-like early transcriptional control sequences. To determine whether the vaccinia transcriptional machinery would recognize these promoters and faithfully transcribe OV genes in vivo the BamHI-E fragment was inserted into the thymidine kinase (TK) locus of vaccinia virus and the recombinant used in transcription studies. Northern blotting analysis of early RNA isolated from 143B-TK- cells infected with the recombinant virus showed that OV genes were transcribed and that the three transcripts of 0.70-(ORF-pp), 0.48- (ORF1), and 0.75-kb (ORF-3) were the same size as their counterparts in OV-infected cells. Analysis of the 5' end of transcripts by S1 nuclease and primer extension showed that the transcriptional start points (tsp) of ORF-pp, ORF-1, and ORF-3 in the recombinant were identical or within four nucleotides of the tsps of the same ORFs in OV. However, there were quantitative differences. ORF-1 was transcribed more efficiently in recombinant virus-infected cells than in those infected with OV and analysis of the putative promoter, 5'-AAAATTGTAAATGTA, showed that it was similar to the 7.5-kDa early promoter of vaccinia virus. This demonstrates that the transcriptional control sequences of OV genes are recognized by vaccinia virus transcriptional factors but that quantitative differences exist suggesting that the generically different transcriptional factors have different promoter sequence requirements for maximal transcription.
...
PMID:In vivo recognition of orf virus early transcriptional promoters in a vaccinia virus recombinant. 154 49

Expression plasmids were constructed in which the human beta-globin gene or a variant of it precisely lacking its two introns was transcribed from its own promoter, the herpes simplex virus type 1 thymidine kinase (HSV-tk) promoter, or the immediate early promoter of human cytomegalovirus (CMV-IE). Forty two hours after transfection of these plasmids into monkey kidney cells, nuclear and cytoplasmic RNA were isolated. Quantitative S1 nuclease mapping and primer extension analysis were used to determine the relative abundances, cellular locations, and leader sizes of the accumulated beta-globin RNAs. Whereas transcripts of all of the intron-containing genes accumulated in the cytoplasm to high levels, transcripts of their cDNA variants were neither stably maintained in the nucleus nor accumulated in the cytoplasm, irrespective of the promoter from which transcription was driven. We conclude that the intron requirement for cytoplasmic accumulation of beta-globin RNA can not be circumvented by synthesis from either the promoter of the intronless HSV-tk gene or the CMV-IE promoter.
...
PMID:Expression from herpesvirus promoters does not relieve the intron requirement for cytoplasmic accumulation of human beta-globin mRNA. 166 15

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.
...
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.
...
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.
...
PMID:Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals. 246 66

The Hinf family is a repetitive nucleotide sequence of the human genome. Certain structural features of Hinf DNA resemble the eukaryotic RNA polymerase II promoters. Therefore, we studied the ability of the Hinf element to function as a transcriptional promoter in mammalian cells. We placed the Hinf element upstream from the thymidine kinase-encoding (tk) sequence in a plasmid construct, pAC401 and introduced it into Ltk- mouse cells. The Hinf-tk plasmid was able to transform Ltk- cells to Tk+ phenotype. In another plasmid construct, pAC Hinf-neo, the Hinf element was inserted upstream from the sequence encoding neomycin (Nm) resistance (neo), and this plasmid was able to confer Nm resistance to HeLa cells. The nature of transcription initiation of the tk gene in four of the Tk+ clones transformed by pAC401 was examined by S1 nuclease analysis, and the transcription start point (tsp) for the tk gene in these clones was mapped within the Hinf element. The same tsp in the Hinf element was found in HeLa cells. Our studies show that the Hinf element functions as a weak promoter.
...
PMID:Localization of a transcription start point within the human Hinf element. 259 45

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.
...
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.
...
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

Enhancers are regulatory DNA elements, usually about 200 base pairs (bp) long, which are able to stimulate transcription of linked genes in eukaryotic cells. This activation can be exerted over large distances, and from a position 5' or 3' to the gene. Enhancers have been identified in viral genomes and cellular genes. Using a transient expression assay, we have analysed transcription of the rabbit beta-globin gene and the thymidine kinase gene from herpes simplex virus with and without a simian virus 40 (SV40) enhancer. S1 nuclease mapping shows a high level of specific transcripts when the genes are linked to the enhancer. To determine whether this increased number of transcripts is due to a higher transcription rate, or perhaps to a shift from nonspecific to specific initiation, we have performed run-on transcription assays with isolated nuclei. Our results, presented here, demonstrate that the SV40 enhancer increases the RNA polymerase density within the linked gene. Therefore, enhancers apparently increase the rate of transcription initiation without influencing the specificity of initiation.
...
PMID:Simian virus 40 enhancer increases RNA polymerase density within the linked gene. 298 14


1 2 3 4 Next >>

---