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)

S1 nuclease isolated from Aspergillus oryzae has been used to investigate the secondary structure of rabbit globin messenger RNA (mRNA). The enzyme, which is specific for single stranded nucleotides, digests globin mRNA to a limited extent, with 65-75% of the mRNA nucleotides resistant to digestion under mild conditions. This limited digestion is not due to enzyme inactivation, but rather to the normal activity of the single-strand nuclease. The reaction was studied as a function of temperature, salt and enzyme concentration. Analysis of the products of digestion on 20% acrylamide- 7M urea slab gels reveals a stable pattern of unique fragments ranging in size from 9 to 71 nucleotides. Separated alpha and beta globin mRNAs show similar, but not identical gel patterns, indicating strong structural similarities between the two species. The high degree of nuclease resistance, along with the fragment patterns seen on polyacrylamide gels, gives evidence to support a model of rabbit globin mRNA which contain specific, rather than random, helical structure.
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PMID:Specific hydrolysis of rabbit globin messenger RNA by S1 nuclease. 90 77

Recent studies indicate that a purified protein, activin A, belongs to the transforming growth factor beta (TGF-beta) superfamily. Similar to TGF-beta, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of alpha, beta, gamma, and epsilon globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for gamma and epsilon globins (209 nucleotides) as well as alpha (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of beta globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic beta-like and alpha globin transcripts also confirmed earlier studies of Northern and slot-blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an epsilon globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells.
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PMID:Regulation of globin gene expression in human K562 cells by recombinant activin A. 173 15

Comparative protein binding studies have been performed on the Xenopus beta globin gene promoter. Erythroblast nuclear extracts 'footprint' over the erythroid-specific consensus sequence, AGGATAAG, which is located immediately upstream of the CCAAT footprint. Nonerythroid cell extracts do not give rise to an AGGATAAG footprint but rather to an extended CCAAT footprint reminiscent of the CCAAT displacement protein (CDP). Erythroblast extracts also protect a sequence similar to the chicken stage selector element (SSE) immediately downstream of the CCAAT box footprint. In contrast to these discrete footprints observed using erythroblast extracts, Xenopus erythrocyte nuclear extracts give rise to more extensive promoter protection. We have previously reported that this promoter is active in transfected HeLa cells when linked to the SV40 enhancer and that transcriptional activation is accompanied by the formation in the chromatin of a nuclease hypersensitive site (HS) in this region. As a first step towards defining the roles of the various promoter-binding proteins in transcriptional activation and HS formation, we transfected deletion mutants of the promoter into HeLa cells. Deletion of the sequences upstream of -116 had no effect on transcription or HS formation. Indeed the upstream boundary of the HS remained unchanged (at around-170) even though plasmid sequences had replaced Xenopus sequences. If the HS boundary reflects resumption of nucleosomal structure, then sequences downstream of -116 must be able to position a nucleosome from at least 50 bp away. beta globin gene activation in a number of transfected cell lines is absolutely dependent on DNA replication. The replication requirement is not a consequence of template copy number or methylation, nor is it dependent on the direction in which the replication fork passes through the gene. We conclude that replication facilitates active transcription complex formation by disrupting a stable association of the template with negative factors, which could include histones. About 200 bp upstream of the Xenopus beta globin gene promoter is a tract of alternating A and T residues which adopts cruciform geometry at low levels of supercoiling. Because of this sensitivity to torsional stress, we have probed the structure of the (AT)n sequence in microinjected Xenopus oocytes, where the Xenopus beta globin gene is transcribed very efficiently. We find that S1 nuclease cleaves specifically in the middle of the (AT)n tract, suggesting that the gene is under torsional stress.
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PMID:Activation mechanisms of the Xenopus beta globin gene. 259 1

K562 cells are induced by hemin to produce gamma and epsilon globin but not beta globin, although the beta globin gene is intact, and when isolated is expressed in a transient expression assay (1, 2). We have previously shown that an epsilon globin gene transferred into K562 cells is expressed and inducible (3). In this paper, we report the stable transfer of a sickle or betaS globin gene into K562 cells. Thirty-six different transformed lines were tested; 24 of 36 lines contained an intact betaS globin gene. However, using S1 nuclease, Dot blot, and Northern blotting analyses, none of these lines showed beta globin mRNA expression. These results indicate that trans acting factors are responsible for the lack of expression of the beta globin gene in K562 cells.
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PMID:Trans acting regulation of beta globin gene expression in erythroleukemia (K562) cells. 299 58

A rapid, sensitive, and specific solution-hybridization method is described that utilizes cloned, double-stranded DNA. The DNA is treated with restriction enzyme(s), and fragments are 32P-labeled at their 5' termini with polynucleotide kinase. A single fragment is partially purified by gel electrophoresis, denatured, and annealed with unlabeled RNA or DNA. The reaction mix is treated with S1 nuclease, precipitated with TCA, and the [32P]DNA counted. Hybridization is recognized only when the 32P label is associated with duplexes that are TCA-precipitable. The specificity of the method was analyzed by annealing experiments with cloned, human globin DNAs. Restriction fragments labeled in the 3' untranslated region of human beta globin clones formed S1-resistant, TCA-precipitable duplexes with beta globin DNA, but not with delta or gamma globin DNA. Thus, a major advantage of the method is that it can distinguish among homologous nucleic acids whose uniformly labeled cDNAs cross hybridize under moderately stringent conditions. This assay is as sensitive as the [3H]cDNA hybridization method, and it circumvents the requirement for purified mRNA as a template for [3H]cDNA synthesis. It also avoids a gel electrophoresis step, it is rapid and quantitative, and many samples can be simultaneously analyzed.
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PMID:Solution hybridization using cloned, 5'-32P-labeled, double-stranded DNA to distinguish among closely related nucleic acids. 628 89

The 5' ends of most normal human beta globin mRNA molecules correspond to a single transcription initiation site, often referred to as the "CAP" site (1-4). Using S1 nuclease mapping and primer extension techniques, we have determined that a minority of beta globin gene transcripts are longer at the 5' ends. These longer molecules comprise about 10% of total beta globin RNA molecules in normal human bone marrow cells and in peripheral blood reticulocytes. The long molecules are transcribed only from the sense strand of DNA and are probably spliced correctly. A DNA segment that includes imperfect "CCAAT" and "TATA" promoter-like sequences begins approximately 150 base pairs (bp) upstream from the normal beta globin gene promoter; this "pseudo-promoter" may function in the initiation of the long globin RNA molecules.
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PMID:A weak upstream promoter gives rise to long human beta-globin RNA molecules. 630 33

A general method is described for the removal of 3'-terminal polyadenylate tracts from eukaryotic messenger RNA to generate essentially homogeneous length products that can be 3'-end labeled and sequenced. Hybridization of specific oligodeoxyribonucleotides was used to direct ribonuclease H to the junction of the 3'-noncoding region and the polyadenylate sequence of rabbit alpha and beta globin mRNAs. Site-specific deadenylylation of both globin mRNAs is demonstrated by partial enzymatic sequence analysis following 3'-terminal labeling with 5'-pCp (where p indicates the labeled phosphate group). The secondary structure of the 3'-noncoding region is studied by enzymatic digestion with S1 nuclease and cobra venom ribonuclease. Structural features of the 3'-noncoding regions of these mRNAs are described, including the protein synthesis termination and the poly(A) addition recognition sites.
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PMID:RNase H-catalyzed site-specific deadenylylation of rabbit alpha- and beta- globin mRNAs. Secondary structure of 3'-noncoding regions. 632 4

For the faithful initiation of transcription of exogenously added fibroin gene, a crude cellular extract was prepared from the posterior silk glands where fibroin gene is specifically transcribed. The faithful initiation was demonstrated by the assay with use of truncated fibroin gene templates and confirmed by nuclease S1 mapping. The transcription initiation of fibroin gene was detected also in the extract from the middle silk glands where fibroin gene is not transcribed, indicating that an expected suppression machinery for the gene was left behind during the extraction procedures. The promoter sequence of fibroin gene required in these homologous extracts (the nucleotide position -29 to +6) is the same as that in a heterologous HeLa cell extract. This result guarantees a limited usefulness of the HeLa cell extract in determining promotor sequence or analyzing general transcription machinery. In the homologous extracts, but not in the heterologous extract, however, it has been shown for the first time in vitro that the 5' flanking sequence upstream from -74 enhances the transcription initiation of fibroin gene. In addition, a remarkable fibroin gene preference in transcription over mouse beta globin and adenovirus 2 major late genes was observed in the homologous extracts. The fibroin gene preference is partly caused by the sequence element upstream from -74 of fibroin DNA.
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PMID:Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5' flanking sequence far upstream. 732 50

We have characterized mRNA expression and transcription of the mouse alpha- and beta-globin loci during development. S1 nuclease and primary transcript in situ hybridization analyses demonstrate that all seven murine globin genes (zeta, alpha1, alpha2, epsilony, betaH1, betamaj, and betamin) are transcribed during primitive erythropoiesis, however transcription of the zeta, epsilony, and betaH1 genes is restricted to the primitive erythroid lineage. Transcription of the betamaj and betamin genes in primitive cells is EKLF-dependent demonstrating EKLF activity in embryonic red cells. Novel kinetic analyses suggest that multigene expression in the beta locus occurs via alternating single-gene transcription whereas coinitiation cannot be ruled out in the alpha locus. Transcriptional activation of the individual murine beta genes in primitive cells correlates inversely with their distance from the locus control region, in contrast with the human beta locus in which the adult genes are only activated in definitive erythroid cells. The results suggest that the multigene expression mechanism of alternating transcription is evolutionarily conserved between mouse and human beta globin loci but that the timing of activation of the adult genes is altered, indicating important fundamental differences in globin gene switching.
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PMID:Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci. 988 4