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)

Synthetic double-stranded DNAs (sDNAs) were prepared from sheep globin mRNA templates isolated from reticulocytes producing either hemoglobin B (HbB) (alpha 2 beta B2), HbC (alpha 2 beta C2), or HbF (alpha 2 gamma 2). These DNAs were inserted into the Eco RI site of plasmid pMB9 by the homopolymer tailing method and used to transform Escherichia coli X1776 to tetracycline resistance. Recombinant clones were identified by colony hybridization and further characterized by molecular hybridization and restriction endonuclease analysis. All plasmids analyzed thus far contained either beta- or gamma-globin DNA sequences. Moreover, sDNAs used for cloning yielded restriction endonuclease fragments consistent with the presence of predominantly beta- or gamma-sDNA, indicating that formation of double-stranded alpha-sDNA proceeds much less efficiently under our conditions than the formation of non-alpha-sDNAs. Three recombinant plasmids, pS beta B2, pS beta C69, and pS gamma 56, were selected for detailed study. These were shown to contain, respectively, beta B-, beta C-, and gamma-DNA sequences by molecular hybridization and by protection of the appropriate cDNAs from S1 nuclease digestion. Each contained all of the restriction endonuclease sites defined for the synthetic sDNAs and protected at least 90% of the sequence length of homologous cDNA. Restriction endonuclease maps of the beta B- and beta C-globin genes were identical at all 12 sites that were mapped, whereas four differences were identified in the gamma gene compared to the two others; three of these corresponded to differences in amino acid sequence of the globins. A method was developed to isolate the anti-mRNA strand of the insert for use as a specific molecular hybridization probe analogous to complementary DNA.
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PMID:Hemoglobin switching in sheep. Synthesis, cloning, and characterization of DNA sequences coding for the beta B, beta C, and gamma-globin mRNAs. 37 94

To date, DNA-binding factors with a developmental pattern of expression have not been described in human erythroid cells to explain the switch from fetal (gamma-) to adult (delta- and beta-) globin gene expression. Here we describe a factor present in nuclear extracts from adult mouse and human hematopoietic cells that binds to an oligopyrimidine repeat approximately 960 base pairs upstream from the human delta-globin gene. The binding site for the factor is within an unusual 250-base-pair domain that is greater than 95% pyrimidines on one strand. This domain is preferentially sensitive to S1 nuclease in supercoiled plasmids, indicating that it can adopt an alternative non-B-DNA conformation. A number of S1-sensitive sites within the domain, including the factor-binding site, have sequence characteristics associated with the formation of a triple helix (H-DNA). The position of the binding site between the fetal and adult beta-globin-like genes, its potential for adopting an unusual secondary structure, and the restricted activity of the binding factor to adult hematopoietic tissues suggest possible roles in hematopoietic cell development and hemoglobin switching.
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PMID:A DNA-binding factor in adult hematopoietic cells interacts with a pyrimidine-rich domain upstream from the human delta-globin gene. 171 93

Bacterial hemoglobin (VtHb) is produced by the gram-negative bacterium, Vitreoscilla, in large quantity in response to hypoxic environmental conditions. The vgb gene coding for VtHb has been cloned in E. coli where it is expressed strongly by its natural promoter. The expression of the vgb gene in Vitreoscilla is transcriptionally regulated by oxygen. When E. coli cells were shifted from 20% to 5% oxygen, vgb specific transcript increased. In E. coli cells with plasmids carrying transcriptional fusions of the vgb gene promoter to either CAT (chloramphenicol acetyl transferase) or xylE (catechol-2,3-dioxygenase) genes, the promoter activity depended on the oxygen level. The concentration of CAT and xylE gene products in cells grown under 5% oxygen was 5-7 times that of aerobically (20% oxygen) grown cells. When the vgb gene promoter was deleted, VtHb was not produced under any conditions. When the promoter was replaced by the E. coli tac promoter, hypoxic oxygen did not affect the level of expression of vgb, but adding IPTG did increase the expression of this gene. These results indicate that the vgb gene promoter is transcriptionally regulated by oxygen even in E. coli, and that microaerobiosis is sufficient to induce vgb expression. The size of S1 nuclease-resistant hybrids, prepared using RNA transcripts protected with restriction enzyme fragments containing the promoter proximal region of vgb, was the same for both Vitreoscilla and E. coli, further evidence that the same promoter is used in both organisms. Transcriptional fusion of the vgb gene promoter to the xylE reporter gene on the broad host range plasmid, pKD-49, was used to demonstrate that the vgb promoter can be expressed in other gram-negative organisms, including Pseudomonas, Azotobacter, and Rhizobium.
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PMID:Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion. 219 33

Processing of the beta major and beta minor globin pre-mRNAs has been compared in murine erythroleukemia cells induced to synthesize hemoglobin by dimethyl sulfoxide or hemin treatment, using both the Northern blot technique and S1 nuclease mapping with 3' and 5' end-labeled probes. The small intervening sequence of both beta-globin pre-mRNAs was removed in one step, although minor amounts of incompletely spliced RNA were detected. During the processing of the large intervening sequence of beta major globin pre-mRNA two internal splice sites were clearly detected. On the contrary, the beta minor globin pre-mRNA did not show any internal splice sites. A model of processing of the mouse adult beta major globin pre-mRNA is proposed.
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PMID:Nuclear precursor molecules of the two beta-globin mRNAs in Friend erythroleukemia cells. 628 40

We have determined the DNA sequence of the 3' end of the putative beta H-globin gene of chickens and used an S1 nuclease protection assay to analyze transcription of this gene. Appearance of mRNA corresponding to the putative beta H-globin gene in the erythroid cells of developing chicken embryos coincides with the reported expression of the beta H-globin protein, a constitutent of hemoglobin H which is a minor component in the definitive cells of late embryos and newly hatched chickens, thereby confirming the tentative identification of this gene as beta H. Analysis of the DNA sequence of the 3'-flanking region of the beta H gene revealed the presence of a complex array of direct repeats. A part of this "repeat cluster" is homologous to an inserted element in the large intron of the goat beta A- and beta C-globin genes.
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PMID:Expression and partial DNA sequence of the chicken beta H-globin gene. 629 82

We studied the abundance and structure of globin mRNAs present in K562 cells both before and after induction of hemoglobin synthesis by hemin. In vitro translation of poly(A)+ RNA from K562 cells generated protein products corresponding to alpha, A gamma-, G gamma-, epsilon-, and zeta-globin mRNAs. Individual globin mRNAs increased 1.5- to 3-fold after induction. Similar results were obtained by measuring steady-state mRNA concentrations of induced and uninduced cells by using S1 nuclease mapping. Globin gene transcripts were correctly initiated and processed. In addition, S1 nuclease analysis revealed the presence of delta-globin mRNA in both control and induced cells. A small percentage of delta-globin transcripts appeared to be initiated upstream from the normal initiation site. beta-Globin mRNA was not detected in any studies. The results (i) suggest that hemin induction of K562 cells is mediated at a transcriptional level and (ii) reveal the dissociation of delta- and beta-globin gene expression in K562 cells compared with normal erythroid cells.
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PMID:Inducible transcription of five globin genes in K562 human leukemia cells. 631 May 80

We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O-thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O-thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5' flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta-mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.
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PMID:Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon. 673 65

Juvenile chronic myelocytic leukemia (JCML) is a rare disorder of early childhood. Characteristic of JCML are the progressive appearance of high levels of fetal hemoglobin (HbF), reflecting a true reversion to a fetal type of erythropoiesis, and the presence of colony-forming cells able to grow in vitro spontaneously in the absence of growth factors. To better understand the relationship between the erythroid abnormalities and the leukemic process, we analyzed the expression pattern of specific genes related to erythroid differentiation--GATA-1, EPOR, alpha-globin, beta-globin, and gamma-globin genes--in JCML peripheral blood (PB) cells and in vitro-derived colonies. Northern blot analysis of PB cells from five JCML patients indicated levels of GATA-1 transcripts much higher than those usually found in other types of leukemic cells, and S1 nuclease protection assay detected significantly increased expression of gamma-globin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single granulocyte-macrophage colony-forming unit (CFU-GM) colonies, obtained in vitro in the absence of added growth factors from four JCML patients, detected GATA-1, EPOR, and globin (alpha and gamma) transcripts in most of the colonies tested, in contrast with control CFU-GM from normal bone marrow, which were positive only for GATA-1. Single JCML colonies were tested for the presence of two different transcripts; whereas alpha- and gamma-globin genes appeared mostly coexpressed, beta-globin mRNA was detected only in a minority of the gamma-globin-positive colonies, indicating that the leukemic pattern of hemoglobin synthesis is mainly fetal. In addition, the leukemic cells occurring during blast crisis of one of our patients displayed the typical features of a stem cell leukemia (CD34+, CD19-, CD2-, myeloperoxidase-). In this sorted CD34+ population, we detected the presence of a marker chromosome, der(12)t(3;12), previously identified in bone marrow cells at diagnosis and an expression pattern superimposable to that of the JCML colonies, consistently displaying a high gamma-globin:beta-globin mRNA ratio. The expression of erythroid markers within populations of leukemic cells, both in vivo and in vitro, supports the hypothesis that abnormal JCML erythroid cells may originate from the same mutated progenitor that sustains the growth of the leukemic cells.
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PMID:Constitutive expression of GATA-1, EPOR, alpha-globin, and gamma-globin genes in myeloid clonogenic cells from juvenile chronic myelocytic leukemia. 779 40