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 cloned the cryptic phn operon of a K-12 strain, phn(EcoK), and analyzed the nucleotide sequence of the phn region (11,672 bp). An mRNA start site upstream of the phnC gene was identified by S1 nuclease mapping. The pho regulon activator PhoB protects a pho box region near the mRNA start in DNase I footprinting and methylation protection experiments. The sequence of the cryptic phn(EcoK) operon was very similar to that of the functional phn operon of an Escherichia coli B strain, phn(EcoB) (C.-M. Chen, Q.-Z. Ye, Z. Zhu, B. L. Wanner, and C. T. Walsh, J. Biol. Chem. 265:4461-4471, 1990). The phnE(EcoK) gene has an 8-bp insertion, absent from the phnE(EcoB) gene, which causes a frameshift mutation. The spontaneous activation of the cryptic phn(EcoK) operon is accompanied by loss of this additional 8-bp insertion. Studies of the structure, regulation, and function of the phn region suggest that the phosphate starvation-inducible phn operon consists of 14 cistrons from phnC to phnP.
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PMID:Molecular analysis of the cryptic and functional phn operons for phosphonate use in Escherichia coli K-12. 184 May 80

The mouse thymidylate synthase (TS; EC 2.1.1.45) mRNA is unusual in that the poly(A) tail is added at the translation stop codon. To determine the sequence requirements for 3' processing of this mRNA, we constructed TS minigenes with deletion and point mutations in potential regulatory sequences. The minigenes were transiently transfected into cultured cells and the effect on 3' processing was determined by S1 nuclease protection assays. These analyses revealed that at least two elements are required for efficient polyadenylylation at the stop codon. The first is an upstream AUUAAA sequence. When this was changed to AUCAAA, polyadenylylation at the stop codon was blocked. However, when it was changed to the canonical AAUAAA hexanucleotide, the amount of TS mRNA increased severalfold. The second element is a stretch of 14 consecutive uridylate residues 32 nucleotides downstream of the stop codon. This U-rich region is absent from the human TS gene, which explains why the human TS mRNA is not polyadenylylated at the stop codon even though the two genes are otherwise almost identical through this region. The most surprising observation was that the U-rich region corresponds to the 3' end of a 360-nucleotide mouse L1 repetitive element that was inserted in opposite orientation to the gene more than 5 million years ago. Thus the polyadenylylation signal of the present mouse TS gene was created by the transposition of a repetitive element downstream of a cryptic polyadenylylation signal.
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PMID:Polyadenylylation signal of the mouse thymidylate synthase gene was created by insertion of an L1 repetitive element downstream of the open reading frame. 215 3

The molecular basis for the defective expression of the mouse class II E beta genes in the H-2w17, H-2q, and H-2f haplotypes has been examined. The results of nuclear run-on transcription and S1 nuclease digestion assays demonstrate that E beta transcription is normal in these haplotypes. Northern blot analyses reveal reduced amounts of E beta RNA of both normal and aberrant size in the w17 and q haplotypes; an even more reduced level of E beta RNA of normal size was detected in the f haplotype. In the preceding study, we reported that the only defect detected in the E beta w17 gene is a single nucleotide insertion in the 5' RNA splice site of the first intervening sequence. S1 nuclease analysis of E beta w17 RNA indicates that splicing at this site is aberrant. One major cryptic RNA splice site is used, leading to reduced amounts of aberrantly processed RNA. Limited use of the mutated splice site and of a second cryptic site also is detected. In all three cases, stop codons in the resulting RNA would prevent their translation. The molecular defect in E beta q appears identical to that of E beta w17. In the f haplotype, even more reduced levels of E beta RNA of both normal and aberrant sizes are found. We thus show that in the three defective E beta alleles, two distinct defects are responsible for the absence of E beta protein synthesis; both of these defects affect RNA processing.
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PMID:Defective E beta expression in three mouse H-2 haplotypes results from aberrant RNA splicing. 246 May 44

A new type of termination signal for RNA polymerase III transcription has been identified. A cloned DNA from salmon highly repetitive sequences, designated as Sm2, serves as a template for an RNA with about 140 nucleotides, but the clone does not have four or more consecutive T residues in the presumed termination site. S1 nuclease mapping analysis clearly demonstrated that termination occurred at the beginning of the AT-rich sequence in the 3' region of Sm2. Studies with a series of 3'-deletion mutants strongly suggested that an AT sequence of more than nine nucleotides functioned as a terminator. Furthermore, results with another series of 3'-deletion mutants showed that a sequence of only nine nucleotides (ATATATATT) in the noncoding strand in fact functioned as a terminator (designated as the 9AT terminator), although with relatively low efficiency compared to a tract of TTTT introduced downstream, and that new cryptic termination sites were introduced in the upstream slight AT-rich region by approach of the 9AT terminator. An AT-rich region downstream from the actual termination site of Sm2 DNA may enhance the efficiency of termination by facilitating detachment of RNA polymerase from the template DNA. Since the nucleotide sequence around the termination site appears to be conserved in the salmon repetitive sequences, this new terminator may be responsible for the generation of a discrete-sized RNA transcribed from salmon total genomic DNA in vitro.
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PMID:Characterization of a new termination signal for RNA polymerase III responsible for generation of a discrete-sized RNA transcribed from salmon total genomic DNA in a HeLa cell extract. 246 46

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

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

S1 nuclease mapping is commonly used to analyze transcription and processing of unlabelled RNAs. However, the S1 protocol that appears best suited to demonstrate splicing of a particular RNA (using an intronless probe that is 5' end-labelled in the downstream exon) is not diagnostic as expected. Rather, both intron-containing RNA and intronless RNA confer protection of probe across the splice juncture. To unambiguously demonstrate correctly spliced RNAs that begin at a specific initiation site, we present a procedure in which unspliced RNA molecules are first cleaved by RNase H following annealing to an intronic DNA fragment and the remaining RNA is then subjected to S1 analysis using an intronless probe present in vast excess. Only spliced, correctly initiated transcripts can protect the probe across the splice junction and up to residue +1. This RNase H/S1 method provides a broadly applicable technique with which to demonstrate splicing and initiation of a variety of transcripts, especially ones from transfected genes that can arise both from the normal and from activated cryptic initiation sites.
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PMID:A combination of RNase H and S1 nuclease circumvents an artefact inherent to conventional S1 analysis of RNA splicing. 303 84

RNA-dependent RNA polymerase activities were detected in purified particles of white clover cryptic viruses 1 and 2. The polymerases of the two viruses had different requirements for optimum activity. Enzyme activity was dependent upon the presence of virus particles, Mg2+, and the four ribonucleoside triphosphates, and was insensitive to actinomycin D, alpha-amanitin, and rifampicin. The labeled reaction products were dsRNAs as indicated by CF 11 column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A and resistance to S1 nuclease. The dsRNAs synthesized in vitro had the same electrophoretic mobilities as the corresponding viral templates.
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PMID:RNA-dependent RNA polymerase activity in two morphologically different white clover cryptic viruses. 335 1

Partially purified carnation cryptic virus (CarCV) preparations possessed RNA-dependent RNA polymerase activity which was absent in comparable preparations from virus-free carnations. Enzyme activity was dependent upon the presence of virus particles, Mg2+, and the four ribonucleoside triphosphates, and was insensitive to inhibitors of DNA-dependent RNA polymerases. The 32P-labeled enzyme reaction products were largely dsRNAs as indicated by resistance to S1 nuclease and RNase A at high but not low ionic strength. The in vitro synthesized dsRNAs hybridized specifically with CarCV genomic dsRNAs, and the radioactive products present in the polymerase reaction mixture sedimented with the virus particles in sucrose density gradients. The data suggest that the RNA-dependent RNA polymerase associated with CarCV particles is a replicase which catalyzes the synthesis of copies of the genomic dsRNAs.
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PMID:In vitro synthesis of double-stranded RNA by carnation cryptic virus-associated RNA-dependent RNA polymerase. 338 65

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.
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PMID:Firefly luciferase gene: structure and expression in mammalian cells. 382 27


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