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)

We report results indicating that expression and hormonally controlled negative regulation of the human pro-opiomelanocortin (POMC) gene in mouse fibroblasts can be accomplished by the placement nearby of a simian virus 40 enhancer sequence. Expression resulting from correctly initiated transcription required the enhancer in cis both in cells stably transfected with the POMC gene and in a transient expression assay with constructs that fused that POMC promoter region to the protein-coding region of the herpes simplex virus thymidine kinase (TK) gene. Negative regulation of POMC transcription by glucocorticoids was demonstrated in transiently infected cells by assaying for TK activity encoded by the POMC-TK fusion constructs and by quantitative S1 nuclease mapping. The sequences responsible for such regulation were shown to be contained within a DNA segment that extends 670 base pairs upstream from the cap site for POMC mRNA.
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PMID:Hormonally mediated negative regulation of human pro-opiomelanocortin gene expression after transfection into mouse L cells. 301 28

A region upstream from the mouse c-mos proto-oncogene, termed upstream mouse sequence (UMS), prevents expression of mos transforming activity. Previous studies suggested that the UMS prevented transcription readthrough. In this study, we constructed a recombinant DNA clone, pHTS3MS, with the UMS inserted downstream from both the mos gene and a truncated long terminal repeat containing only the U3 enhancer region. In this position UMS did not inhibit mos transforming activity. We examined cells transformed by pHTS3MS for RNA expression. S1 nuclease analysis showed that the UMS provides two polyadenylation signals to mos-containing RNA and nuclear run-on transcription showed that the primary transcripts terminate in UMS. In addition, using portions of the UMS, we found that a 360-bp fragment containing the UMS polyadenylation signals and sites inserted between the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (tk) and its promoter inhibits tk transforming activity by 99% and prevents detectable expression of this construct in transient expression assays. Thus, the UMS must contain signals for polyadenylation and appears to function as a transcription terminator.
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PMID:Sequences upstream from the mouse c-mos oncogene may function as a transcription termination signal. 301 57

The herpes simplex virus (HSV) type 1 thymidine kinase gene (tk) was resected from its 3' end with BAL 31 exonuclease. Two sets of plasmids were isolated that lacked information distal to the two copies of the hexanucleotide 5'-AATAAA-3' located at the 3' end of the HSV tk gene. The presence of a simian virus 40 origin of DNA replication in each plasmid facilitated analysis of patterns of transcription in transfected Cos-1 monkey cells. Transcription analyses were performed with an S1 nuclease protection assay. Efficient processing and polyadenylation at the normal site still occurred when all sequences more than 44 or 46 base pairs (bp) downstream from the first AATAAA were removed (pTK311R/SV010 and pTK209R/SV010). Removal of an additional 7 bp (pTK312R/SV010) decreased the amount of tk mRNA processed at that normal site, and tk mRNA polyadenylated at a cryptic site within pBR322 sequences began to appear. The normal processing and polyadenylation site was not used at all when an additional 12 bp was removed (pTK314R/SV010); the small amount of tk mRNA produced was processed and polyadenylated at the cryptic pBR322 site. The region of the tk gene critical for efficient processing and polyadenylation of tk mRNA is located 20 to 38 bp downstream from the first AATAAA, distal to the polyadenylation site, and as RNA can form a stem-loop structure containing AAUAAA. Similar G + T-rich elements were located in DNA fragments which substitute efficiently for the HSV tk processing and polyadenylation signal and were not found in AATAAA-containing DNA fragments which substitute inefficiently for the HSV tk signal.
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PMID:Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation. 301 51

Transcription of mouse mammary tumor virus DNA is stimulated by steroid hormones. The DNA sequences involved in this regulation are located in the viral long terminal repeat between positions -200 and -50 with respect to the transcription initiation site. In this region four, one distal and three proximal, in vitro binding sites for the glucocorticoid hormone-receptor complexes have been identified. We have prepared a series of 5' and 3' deletions of this region, using the exonuclease ExoIII. Combination of suitable 5' and 3' fragments enabled us to reconstitute the entire long terminal repeat with small internal deletions. The mutated long terminal repeats linked to the coding region of the Herpes simplex virus thymidine kinase gene were introduced into LTK- aprt- cells by transfection. Transcription from the mouse mammary tumor virus promoter in the presence or absence of hormone was assayed by nuclease S1 mapping. Deletion of the proximal in vitro binding sites resulted in a decrease in hormonal inducibility. When a synthetic oligonucleotide harboring the sequence of the distal in vitro binding site was inserted at the site of the proximal ones, hormone response was restored. This indicated that the distal binding site can replace the proximal ones in their hormone-regulatory function. However, insertion at the same site of an oligonucleotide containing the sequence 5' TGTTCT 3' found in all four binding sites, did not restore the hormone response, indicating that sequences flanking the TGTTCT motif are required for hormone response. Insertion of an unrelated DNA fragment at the site of the proximal binding element deletion completely abolished the hormone response. Analyses of different proximal binding-site deletion and insertion mutants suggested the presence of a transcriptional element located downstream from the most proximal hormone-receptor binding site.
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PMID:Functional analysis of the glucocorticoid regulatory elements present in the mouse mammary tumor virus long terminal repeat. A synthetic distal binding site can replace the proximal binding domain. 302 40

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

A vaccinia virus (VV) gene required for DNA replication has been mapped to the left side of the 16-kilobase (kb) VV HindIII D DNA fragment by marker rescue of a DNA- temperature-sensitive mutant, ts17, using cloned fragments of the viral genome. The region of VV DNA containing the ts17 locus (3.6 kb) was sequenced. This nucleotide sequence contains one complete open reading frame (ORF) and two incomplete ORFs reading from left to right. Analysis of this region at early times revealed that transcription from the incomplete upstream ORF terminates coincidentally with the complete ORF encoding the ts17 gene product, which is directly downstream. The predicted proteins encoded by this region correlate well with polypeptides mapped by in vitro translation of hybrid-selected early mRNA. The nucleotide sequences of a 1.3-kb BglII fragment derived from ts17 and from two ts17 revertants were also determined, and the nature of the ts17 mutation was identified. S1 nuclease protection studies were carried out to determine the 5' and 3' ends of the transcripts and to examine the kinetics of expression of the ts17 gene during viral infection. The ts17 transcript is present at both early and late times postinfection, indicating that this gene is constitutively expressed. Surprisingly, the transcriptional start throughout infection occurs at the proposed late regulatory element TAA, which immediately precedes the putative initiation codon ATG. Although the biological activity of the ts17-encoded polypeptide was not identified, it was noted that in ts17-infected cells, expression of a nonlinked VV immediate-early gene (thymidine kinase) was deregulated at the nonpermissive temperature. This result may indicate that the ts17 gene product is functionally required at an early step of the VV replicative cycle.
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PMID:Nucleotide sequence and transcript organization of a region of the vaccinia virus genome which encodes a constitutively expressed gene required for DNA replication. 303 68

Hybrid genes containing mRNA encoding sequences for herpes virus thymidine kinase (tk), chloramphenicol acetyltransferase (CAT), or Drosophila alcohol dehydrogenase (Adh), ligated to truncated Drosophila melanogaster heat-shock protein 70 (hsp 70) gene promoters or to synthetic sequences containing one or several copies of a previously defined heat-shock consensus sequence, were transfected into cultured Drosophila line S3 cells. Each construction was then assayed for gene expression at 25 degrees C and 37 degrees C, using a CAT enzyme assay, slot blot hybridization, or S1 nuclease protection analysis. In the Drosophila cell transient expression assay system, we found that deletions extending beyond position -97, or synthetic constructions containing a single heat shock consensus sequence, were not induced by high-temperature shock. In constructions containing deletions extending to position -186, -130, or -97, in the hsp 70 promoter, and in synthetic constructions containing tandemly spaced heat-shock consensus sequences mRNA transcription was greatly induced by high temperature.
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PMID:Natural and synthetic heat shock protein gene promoters assayed in Drosophila cells. 309 68

We have reported the isolation of cis-acting regulatory DNA sequences promoting expression of the herpes virus thymidine kinase gene in vaccinia virus recombinants. In this work we show that each of the inserts from recombinants VpT25, 28, 36 and 56 contains a vaccinia virus early promoter. The position of each of the early RNA start sites in the nucleotide sequence of these four vaccinia virus inserts was precisely mapped by an S1 nuclease mapping procedure. Among the four recombinants analysed only VpT56-infected cells also contained a substantial amount of a transcript with the same 5' end at late period. The insert present in VpT25 contained a new late RNA start site 50 nucleotides upstream from that of the early RNA. The four inserts were mapped on the vaccinia virus genome. We also localized the 5' end of the mRNA of a vaccinia virus host-range gene, whose DNA nucleotide sequence has recently been established. The 45 nucleotides preceding the RNA start site from most of 19 known vaccinia virus early promoters were found to be A + T-rich (at least 80%) and contained shorter A-rich (at least 60%) regions, beginning approximately 25 nucleotides upstream from the RNA start site. The information content, as expressed by the parameter Rsequence, of early vaccinia virus promoters revealed ten bits of information in the sequence of 28 nucleotides upstream from the early RNA start sites. Most of the information needed to locate an early promoter is contained within the nucleotide sequence upstream from an RNA start site. A consensus sequence consists of two blocks: the sequence AA(A/T)N(T/A)N(A/G)AAAANAANA starting at position -27 and the sequence (T/A)(C/T)N(A/T)T(A/G) starting at position -5. It was concluded that vaccinia virus early promoters may be characterized by an A + T-rich region of approximately 45 nucleotides preceding the RNA start site and include a specific 3'-terminal sequence of 28 nucleotides containing at least ten bits of information. A procedure for localizing putative early RNA start sites in nucleotide sequences is proposed.
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PMID:Characterization of vaccinia virus early promoters and evaluation of their informational content. 343 Jun 23

A plasmid containing 1.8 kilobase pairs of rat growth hormone (rGH) promoter and upstream flanking sequences fused to the bacterial gene for chloramphenicol acetyltransferase (CAT) was transiently introduced into pituitary, fibroblast, and kidney cell lines. Significant CAT activity was detectable only in the pituitary cell lines, demonstrating that this relatively large fragment directs strongly cell-type-specific expression. However, plasmids containing only 200-300 bases of rGH promoter and flanking sequences directed expression of CAT in all three cell types, suggesting that upstream sequences directly repress the activity of a minimal rGH promoter in nonpituitary cell types. S1 nuclease analysis showed that the RNA synthesis directed by one of the short rGH promoter fragments in fibroblasts initiated from the site used by the natural promoter in pituitary cells. Insertion of rGH upstream sequences in their natural orientation upstream of the mouse metallothionein I promoter caused a decrease in its activity in fibroblasts by a factor of 4, but there was a 2.5-fold increase in its activity in pituitary cells. Insertion of the rGH fragment upstream of the thymidine kinase promoter in either orientation lowered its activity in both fibroblasts and pituitary cells. Thus, the negatively acting rGH flanking sequences can act on a heterologous promoter and have at least some of the properties of positively acting enhancers.
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PMID:Repression mediates cell-type-specific expression of the rat growth hormone gene. 346 54

A vaccinia virus late gene coding for a major structural polypeptide of 11 kDa was sequenced. Although the 5' flanking gene region is very A+T rich, it shows little homology either to the corresponding region of vaccinia early genes or to consensus sequences characteristic of most eukaryotic genes. Three DNA fragments (100, 200, and 500 base pairs, respectively), derived from the flanking region and including the late gene mRNA start site, were inserted into the coding sequence of the vaccinia virus thymidine kinase (TK) early gene by homologous in vivo recombination. Recombinants were selected on the basis of their TK- phenotype. Cells were infected with the recombinant viruses and RNA was isolated at 1-hr intervals. Transcripts initiating either from the TK early promoter, or from the late gene promoter at its authentic position, or from the translocated late gene promoters within the early gene were detected by nuclease S1 mapping. Early after infection, only transcripts from the TK early promoter were detected. Later in infection, however, transcripts were also initiated from the translocated late promoters. This RNA appeared at the same time and in similar quantities as the RNA from the late promoter at its authentic position. No quantitative differences in promoter efficiency between the 100-, 200-, and 500-base-pair insertions were observed. We conclude that all necessary signals for correct regulation of late-gene expression reside within only 100 base pairs of 5' flanking sequence.
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PMID:One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. 385 86


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