Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.21.1 (DNase)
 7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Liposomes entrapping plasmids pSV2-neo DNA, pUC18-ras DNA, pSV2-neo-ras DNA and linear DNA were prepared. The liposomes were composed of DOPC/Chol/OA (4:4:3) and pH-sensitive DOPE/Chol/OA (4:4:3), respectively. The efficiency of DNA entrapment was about 50%. Gel electrophoresis analysis showed: Liposome-entrapped DNA was not digested by DNase; The entrapped DNA molecules were intact and stable for at least 5-6 months at 4 degrees C. During preparation of pH-sensitive liposome, the pH must be kept at 8.0.
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PMID:[Preparation of liposomes entrapping plasmid DNA and linear DNA]. 139 41

Overexpression of ras proto-oncogenes has been implicated in cancer development. We therefore initiated a study of the human N-ras promoter to determine the regions that control N-ras expression and their potential for interaction with DNA-binding proteins. N-ras CAT constructs were stably integrated into K562 cells by electric field-mediated gene transfer in order to determine functional regions within the human N-ras promoter. A significant proportion of promoter activity was found to lie within a 439 bp fragment comprising an untranslated exon (exon 1) with the adjacent 5' sequence and a small CpG island. A 109 bp [corrected] fragment at the 5' end of exon 1 was essential for promoter activity, while a 45 bp [corrected] deletion from within this region decreased promoter activity by two-thirds. Unlike the human H-ras and mouse K-ras promoters, the N-ras promoter did not exhibit bidirectional activity. DNAse footprinting of the 439 bp fragment revealed seven protected regions, many of which contain sequences homologous to known DNA-binding protein sites (MLTF/myc, CREB/ATF, AP-1, AP-2, myb and E4TF1). In contrast, four putative Sp1 sites did not footprint. Using purified MLTF and appropriate competitors in gel shift and DNAase footprinting assays, we demonstrated binding of MLTF to the MLTF consensus sequence within exon 1.
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PMID:Characterization of the human N-ras promoter region. 157 Jan 52

To gain insight into the normal controls mediating expression of the c-Ki-ras protooncogene, we have identified DNA sequence elements within its promoter that are essential for transcriptional activity. Transient expression assays using the bacterial chloramphenicol acetyltransferase gene were used initially to localize regions directing primary promoter function. Stepwise deletion of 5' promoter sequences resulted in a gradual decrease in the ability to drive transcription of the reporter gene, suggesting that this promoter is composed of multiple cis-acting elements. Gel mobility-shift and DNase protection studies involving a 166-base-pair DNA fragment allowed the identification of protein-binding sites corresponding to these multiple regulatory elements. One element demonstrating particular transcriptional influence exists as a homopurine/homopyrimidine-rich region that in vitro exhibits S1 nuclease sensitivity and binds at least one nuclear protein. Data from competition binding experiments suggest that this nuclear factor may be influential in the regulation of other essential growth-control genes as well.
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PMID:An S1 nuclease-sensitive homopurine/homopyrimidine domain in the c-Ki-ras promoter interacts with a nuclear factor. 218 46

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, whereas the paternal C3H/He strain and the maternal C57BL/6 strain exhibit a 60% and a negligible incidence, respectively. In addition, both male and female B6C3F1 mice are extremely sensitive to chemical induction of hepatocarcinogenesis. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently, Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors. The objective of this study was to examine a possible point of transcriptional control of Ha-ras, Ki-ras, and myc in all three mouse strains, our hypothesis being that these oncogenes may be primed for expression in the nascent liver of those strains exhibiting a high spontaneous hepatoma incidence. A positive correlation has been established between gene expression and the presence of DNAase I hypersensitive sites. DNase I hypersensitive sites were observed in the Ha-ras and myc oncogenes in the three mouse strains. However, Ha-ras appears to possess an additional site in B6C3F1 and C3H/He as compared to C57BL/6. Similarly, the Ki-ras oncogene exhibited a DNase I hypersensitive site only in B6C3F1 and C3H/He mouse liver. These results indicate that the hepatoma-prone strains (B6C3F1 and C3H/He) may have a greater potential for Ha- and Ki-ras expression than does the non-hepatoma-prone strain (C57BL/6).
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PMID:Differential DNase I hypersensitivity of ras oncogenes in B6C3F1, C3H/He, and C57BL/6 mouse liver. 248 56

Members of the ras gene family encode proteins that when overproduced or mutated can transform immortalized mammalian cells. It is therefore important to understand the mechanisms by which the ras genes are regulated. The promoter region of the human Harvey ras proto-oncogene c-Ha-ras1 initiates RNA transcription at multiple sites and contains repeated copies of the hexanucleotide GGGCGG and its inverted complement CCGCCC, referred to as GC boxes. These GC boxes consist of sequences identical to those found in the SV40 early promoter, where the human cellular transcriptional factor Sp1 binds. Footprinting analysis with deoxyribonuclease I was used to show that Sp1 binds to six GC box sequences within the c-Ha-ras1 promoter. An in vivo transfection assay showed competition between the 21-base pair repeats of the SV40 promoter and the c-Ha-ras1 promoter for common regulatory factors. In this system the presence of Sp1 is apparently required for c-Ha-ras1 transcription. Analysis of deletions of the c-Ha-ras1 promoter region by means of a transient expression assay revealed that the three Sp1 binding sites closest to the RNA start sites were sufficient for full transcriptional activity.
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PMID:Binding of the Sp1 transcription factor by the human Harvey ras1 proto-oncogene promoter. 301 74

The nucleotide sequence of the 5' end distal region of the human c-K-ras gene promoter was determined. This region, coincident with a variable DNAse I hypersensitive site in native chromatin, contains sequence similarities with known enhancers. A 400 bp MstII DNA fragment of this region stimulated in cis the correctly initiated transcription of the human beta-globin gene in transfected Hela cells. The stimulation of beta-globin transcription (5-6 fold) was dependent on the distance and orientation of the c-K-ras sequences and on the presence of the CCAAT and CACCC elements in the beta-globin promoter. Interaction of nuclear factors with these c-K-ras sequences was analysed by DNAase I footprinting assays using Hela nuclear extracts. A protein binding to these sequences was identified as nuclear factor 1 (NF-1) by DNAase I competition footprinting experiments. However, disruption of the c-K-ras NF-1 binding site by insertion mutagenesis had no effect on the transcriptional activity of the c-K-ras element.
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PMID:Initial characterization of a potential transcriptional enhancer for the human c-K-ras gene. 328 54

The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment of HCV infection.
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PMID:Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody. 751 28

We have cloned a 1.8-kb segment of DNA just adjacent to the 5' end of the 6.4-kb BamHI fragment which contains the Ha-ras oncogene (ras1; GenBank notation HUMRASH). Electrophoretic mobility shift assays (EMSA) indicate the presence of multiple nuclear protein-binding sites within the 1.8-kb segment. At least three putative regulatory protein-binding sites have been identified in a 206-bp subfragment by DNase I footprint analysis. Within the subfragment containing the DNase-protected regions, there is also a segment containing a number of consensus sequences for DNA-binding proteins and two sub-sequences that exhibit strong sequence homology to at least two previously characterized enhancers. These data suggest that a novel set of regulatory elements may lie as far as 2 kb upstream from the normal Ha-ras transcription start points.
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PMID:Characterization of a 1.8-kb DNA segment located upstream from the human Ha-ras protooncogene and possibly regulating its function. 772 Nov

The Ha-ras gene is one of the three oncogenes (Ha-ras, Ki-ras, and N-ras) of the ras superfamily of small G proteins. The p21ras proteins encoded by the ras genes are key proteins involved in the transduction of signals from membrane receptor-tyrosine kinases to downstream targets. The ras genes seem to play a ubiquitous role in the control of cell proliferation and cell differentiation. At the same time, ras genes may perform specific differentiated functions in certain cell types. Little is known about the regulation of expression of the Ha-ras gene. The first intron of the Ha-ras gene has been reported to be highly conserved between human and rodent. We investigated the role that this intron may play in the regulation of expression of Ha-ras. The promoter region of the Ha-ras gene exhibits characteristics of a housekeeping gene. Deletion analysis shows the existence of an enhancer-type element in the 5' region of the first intron (intron 0). DNase 1 footprinting experiments reveal five sites that interact with nuclear proteins from fibroblast and epithelial cell lines. Deletion and site-directed mutagenesis of three of these sites show that two are involved in a positive effect and one in a negative effect on the regulation of expression of the mouse Ha-ras gene.
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PMID:Regulatory elements in the first intron of the mouse Ha-ras gene. 789 67

Lysyl oxidase is an extracellular enzyme involved in connective tissue maturation that also acts as a phenotypic suppressor of the ras oncogene. To understand how this suppressor is controlled, gene transcription was studied and the promoter was characterized. Nuclear runoff transcription assays indicated that the markedly reduced amounts of lysyl oxidase message detected after ras transformation resulted from inhibition of lysyl oxidase transcription. Interferon-mediated phenotypic reversion of ras transformed cells, in which the ras oncogene continued to be expressed, was accompanied by the restoration of lysyl oxidase transcription. Reporter gene assay of a transfected mouse lysyl oxidase promoter indicated that it was active in the transformed background, despite the silencing of the endogenous lysyl oxidase promoter. The detection of comparable amounts of mRNA for transcription factors IRF-1 and IRF-2 in normal and ras-transformed cell lines suggests that the differential transcription of lysyl oxidase was not due to regulation of IRFs. Lysyl oxidase promoter activity was localized to a 126 bp region that includes two consensus TATA boxes with associated confirmed cap signals. Analysis of a human lysyl oxidase promoter sequence indicated similar promoter elements and extensive sequence identity with the mouse promoter. The binding of transcription factor AP2 to sites predicted in the control region was confirmed by DNase footprinting. Lysyl oxidase transcription was stimulated by dexamethasone treatment of cells, but this effect could not be assigned within the approximately 3 kb region tested in reporter gene constructs. The promoter activity of the lysyl oxidase reporter gene construct was completely abolished by in vitro DNA methylation, suggesting that the transcriptional suppression after transformation by the ras oncogene may involve DNA methylation.
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PMID:Epigenetic inhibition of lysyl oxidase transcription after transformation by ras oncogene. 1039 Nov 27


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