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)

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

We report the isolation and complete sequence of the gene encoding the rabbit erythroid-cell-specific 15-lipoxygenase (RBC 15-LOX), containing 14 exons spanning 8.0 kb. The transcription start point was mapped by S1 nuclease-protection experiments and comparison with the sequence of the RBC 15-LOX mRNA, as defined previously by primer extension experiments. The promoter contains a TATA-like motif, but no CCAAT motif in the canonical position, and lies within a 'CpG-rich island'. Functional analysis of the immediate 5'-flanking DNA by transfection experiments shows that a 150 nucleotide (nt) 5' fragment linked to the chloramphenicol acetyltransferase gene acts as a functional promoter in both erythroid and nonerythroid cell lines and responds in an erythroid-specific manner to the enhancer from the Friend murine leukaemia virus long terminal repeat, whereas a 40-nt fragment is inactive. Intron 7 contains eight copies of a 54-nt repeat containing a region with homology to the simian virus 40/immunoglobulin gene enhancers.
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PMID:The promoter structure and complete sequence of the gene encoding the rabbit erythroid cell-specific 15-lipoxygenase. 261 16

The enzyme 5-aminolevulinate synthase (ALA-S) catalyzes the first step in heme biosynthesis. In this study, the mouse erythroid gene has been cloned and analyzed in order to investigate the regulation of ALA-S expression during erythroid differentiation. The gene spans approximately kbp and consists of 11 exons and 10 introns. The first exon is 37 bp, non-coding, and followed by a 6kb intron. The mRNA capsite was mapped by primer extension and defines a promoter that contains no apparent TATA element. S1 nuclease analysis detects the presence at low levels of a 45 bp-deleted form of the ALA-S mRNA created by the use of an alternative splice site at the intron 2/exon 3 junction. Five DNAse I hypersensitive sites were detected in chromatin from uninduced and induced MEL cells. One site is at the promoter; the others are in the body of the gene. No significant differences were observed in the patterns or intensity of the hypersensitive sites in the uninduced and induced MEL cells, however, no sites in ALA-S were observed in NIH 3T3 cells or in deproteinized DNA. Thus, these sites are specific for erythroid chromatin but appear to be established at an earlier stage of differentiation than represented by the uninduced MEL cell.
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PMID:Structure of a mouse erythroid 5-aminolevulinate synthase gene and mapping of erythroid-specific DNAse I hypersensitive sites. 278 Mar 18

Previous studies have indicated that control and hemin-treated human erythroleukemia K-562 cells fail to produce adult-type beta-globin mRNA transcripts and to translate them into nascent beta-globin chains. Expression of the beta-globin DNA sequences in K-562 cells can occur, however, under certain conditions. To readdress this issue and to examine the possibility of whether these cells produce immature and untranslatable beta-globin RNA transcripts, we prepared total cytoplasmic RNA from control and inducer-treated cells and performed Northern blot hybridization analysis using 5' end-labeled fragments of the human beta-globin DNA rather than 3' end fragments as probes. Although hybridization of both cytoplasmic and nuclear K-562 RNA with a 32P-labeled 3' end fragment (1.6kb Bam H1 cut) coding for a large part of the first exon of beta-globin failed to detect beta-globin RNA transcripts, hybridization with a 5' end 32P-Labeled 2.0kb Bam H1 fragment (coding for the third exon and part of the second) revealed the presence of relatively small (less than 7S) RNA molecules both in nuclear and cytoplasmic fraction. S1 nuclease mapping of both cytoplasmic and nuclear RNA with the use of 5' end-labeled 2.0 kb Bam H1 fragment of human beta-globin DNA indicated protection of a small portion located 64 bp 5' upstream from the Bam H1 site of the second exon. The amount of protected portion was relatively higher in K-562 cells undergoing erythroid maturation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hemin increase production of beta-like globin RNA transcripts in human erythroleukemia K-562 cells. 279 52

Using a restriction enzyme accessibility assay, we have previously demonstrated that the chromatin structure immediately proximal to the goat beta F-, beta C-, and beta A-globin genes changes in a manner which parallels their developmentally regulated expression. More specifically, the PvuII recognition sequence, located 9 nucleotides upstream from the transcriptional start site in each of the three genes, is accessible to digestion only in nuclei prepared from erythroid tissue in which the respective gene product is expressed. Here we describe two restriction enzyme sites further upstream from the transcription start sites (HindIII at -700 and SacI at -480) which were not accessible to digestion in fetal erythroid nuclei. Conversely, two sites within the second coding block of the beta F gene (AccI at +276 and BamHI at +470) were accessible in fetal erythroid tissue. The corresponding sites in the beta C and beta A genes were not available for digestion in the same fetal tissue. Processive exonuclease III digestion in situ from the three accessible restriction enzyme sites in the beta F gene allowed us to define more closely the limits of these open regions. Resistance to exonuclease III digestion was encountered at or near both intron-exon junctions flanking the first intervening sequence of the beta F gene. Conversely, no resistance to exonuclease III digestion was evident in either the first or second coding blocks or the 5' untranslated region. Digestion upstream from the PvuII site of the beta F gene was negligible. High-resolution mapping by S1 nuclease analysis indicated that the endpoint of exonuclease III digestion from this site lay immediately downstream of the ATA box.
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PMID:Chromatin fine-structure mapping of the goat beta F gene in fetal erythroid tissue. 282 4

We have analyzed the chromatin structure of a region that encompasses 14.4 X 10(3) base-pairs of the chicken histone H5 locus in adult erythroid cells at different stages of maturation. Seven of eight major lineage-specific DNase I-hypersensitive sites, some of which show complex substructure, were found in the flanking regions of the gene. The hypersensitivity of some of these sites is modulated during erythrocyte maturation in a way that parallels the transcriptional activity of the gene. DNase I, micrococcal nuclease, and S1 nuclease recognize the same regions, which differ from those cleaved by S1 on supercoiled plasmid DNA. This suggests that hypersensitivity of DNA in chromatin reflects a greater accessibility of the DNA rather than its altered conformation. The DNA sequence of some of the DNase I target sites contains repeated motifs, (T-C-C-C)2, (T-C-C)2, (T-G-G-G-G)2, which are found in the hypersensitive sites of other genes. Detailed analysis across sections of the H5 gene and flanking sequences revealed differences in the DNase I sensitivity of the different regions examined. Notably, the first one-third of the gene is more sensitive than the rest. The sequences downstream from the region where most RNA polymerases terminate transcription were found to be the most resistant.
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PMID:Fine analysis of the active H5 gene chromatin of chicken erythroid cells at different stages of differentiation. 302 21

In order to test whether the ontogenetic origin of human parental cells influences the expression of globin genes, we did fusions of mouse erythroleukemia (MEL) cells with fetal or adult nonerythroid cells and we assessed globin expression in hybrids. For selection of hybrid clones we used a monoclonal antibody that recognizes a human chromosome 11-linked cell surface marker. Globin expression was assessed with fluorescent antibody labeling, globin isoelectric focusing, and S1 nuclease mapping. Chromosome 11-containing hybrids from human nonerythroid cells (adult or fetal lymphoid cells; fetal fibroblasts) produced human adult (beta) but not fetal (gamma) globin chains. These results suggest that the MEL x nonerythroid cell hybrids express the adult human globin independent of whether the human cells derive from the fetal or from the adult stage of human development. Our findings, together with previous observations in MEL x fetal erythroid or MEL x HEL hybrids, show that the globin program of the parental human cell is the main determinant of the expression of fetal human globin in these hybrids.
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PMID:Only adult hemoglobin is produced in fetal nonerythroid x MEL cell hybrids. 346 79

Lepore globin is synthesized in markedly diminished amounts (approximately 10% to 15% of normal beta-globin) in human erythroid cells. To study the molecular mechanisms responsible for the diminished biosynthesis of Lepore globin, the Lepore-Boston gene was cloned from a charon phage DNA library and expressed in HeLa cells. Northern blotting and S1 nuclease analyses indicated that the Lepore gene produced less globin mRNA than a beta-gene and more than a delta-gene. The results indicate that expression of the Lepore-Boston gene in HeLa cells is reduced to an extent comparable to that seen in erythroid precursors in vivo. This indicates that the decrease in Lepore globin gene transcription is due to the delta-nucleotide sequences either in the 5' flanking region or within this gene.
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PMID:Expression of a cloned Lepore globin gene. 394 May 44

We have attempted to investigate the conformational basis and developmental relevance of globin-associated hypersensitive sites in avian and human cells. Our results indicate that once formed, DNaseI-hypersensitive sites have the capacity to propagate their own structure to daughter cells in the absence of the original events that have led to their formation. We postulate that the single-stranded character (as revealed by S1 nuclease) of these DNaseI-hypersensitivity sites could explain these results. In addition, we show that the locations of hypersensitive sites in an active avian endogenous retrovirus remain unmethylated in mature sperm and suggest that this phenomenon could lead to the propagation of structural information from germ line to fertilized egg. We have also investigated the chromatin structure of the chromosomal DNA regions containing the human G gamma-, A gamma-, delta-, and beta-globin structural genes in both fetal and adult erythropoietic tissues. Our results indicate that DNaseI introduces specific cuts into the beta-globin gene cluster in erythroid cells, but not in white blood cells. The predominant sites are located at the 5' sides of the G gamma-, A gamma-, delta-, and beta-globin genes, within 200 bp of the respective CAP sites. Examination of fetal liver cells has revealed the presence of hypersensitive sites at the 5' side of all four genes, whereas analysis of adult bone marrow has revealed the characteristic sites near the delta- and beta-globin genes but no hypersensitive sites at the 5' termini of the G gamma- or A gamma-globin genes. The presence of delta- and beta-hypersensitive sites in fetal cells suggests that the increment in expression of the delta and beta genes during development most likely involves the modulation of another pathway to gene expression.
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PMID:Developmental aspects of chromatin structure and gene expression. 619 53

The K562 human leukemia cell is an erythroid-like cell that may serve as a model for the study of globin gene expression in transcriptionally active human erythroid cells. K562 cells express all globin genes with the exception of that for beta-globin; failure to produce beta-globin could result from an acquired mutation in each of the beta-globin genes or from an alteration in the regulatory factor environment of the beta-globin gene. To uncover a possible acquired mutation, restriction endonuclease analysis of genomic K562 DNA and expression studies of a cloned K562 beta-globin gene were carried out. Restriction endonuclease analysis revealed no structural alteration of the K562 beta-globin genes. Analysis of the polymorphic Ava II site in intervening sequence 2 of the beta-globin gene showed that K562 cells contain two different beta-globin alleles, both of which are inactive. A K562 beta-globin gene was cloned, ligated into the expression vector pLTN3B, and introduced into COS cells. Transcripts were analyzed by RNA blot, dot blot, S1 nuclease mapping, and primer extension assay. The cloned K562 beta-globin gene was transcribed in COS cells as efficiently as a normal beta-globin gene introduced into COS cells; the mRNA was 10 S and polyadenylylated; the 5' and 3' termini and the processing of transcripts were identical to that of mRNA transcribed from a normal gene. Based on these data we suggest that the absence of beta-globin gene expression results not from an alteration in the beta-globin gene, but from a quantitative or qualitative alteration in a trans-acting factor important in beta-globin gene expression.
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PMID:A beta-globin gene, inactive in the K562 leukemic cell, functions normally in a heterologous expression system. 620 98


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