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)

Changes in insulin-regulated gene expression occur in a time- and tissue-dependent fashion. To monitor these changes we have adapted the S1 nuclease protection assay to allow simultaneous estimation of multiple RNA species in a single sample by using synthetic oligonucleotides of various lengths as probes for specific RNA species, which can then be resolved by electrophoresis. The multiple S1 nuclease protection assay was used to assess the influence of insulin on the RNA concentrations of 12 different genes in human skeletal muscle. Estimates obtained by this assay were comparable with those obtained by Northern analysis. RNA levels for proto-oncogene c-src displayed a transient 4-fold increase, whereas RNA levels for type 1 protein phosphatase were suppressed by 50% during the same time period. RNAs corresponding to known insulin-responsive genes such as c-fos, c-myc, c-Ha-ras, and c-src displayed rapid and transient 2-4-fold increases between 30 and 60 min as detected by either Northern analysis or the multiple S1 nuclease protection assay. In addition, RNA levels for the insulin receptor, Glut-4, Glut-3, and c-jun were apparently unaffected by exposure of the cells to insulin.
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PMID:Use of a multiple S1 nuclease protection assay to monitor changes in RNA levels for type 1 phosphatase and several proto-oncogenes in response to insulin. 137 96

The importance of the R region in basal human immunodeficiency virus type 1 (HIV-1) transcription was addressed by comparing a panel of HIV-1 R region mutants using in vitro and in vivo assays. Using deletion, base substitution mutants, and compensatory mutants, the precise R region sequences essential for basal HIV-1 promoter activity in vitro were mapped to sequences between +17 to +21. Within this regulatory domain, nucleotides +19 and +21 appear to be critical. The effect of these mutations on steady state RNA levels in transfected cells has been analyzed by S1 nuclease protection assay using uniformly labeled probes. Two main conclusions may be drawn from these studies. First, HIV-1 basal transcription is abundant, with the majority of correctly initiated transcripts truncated between sequences +57 to +70. Second, analysis of the compensatory mutants indicates the secondary structure of the nascent R region RNA is not an obligate requirement for the production of the truncated transcripts. Mutations in R region primary sequence that selectively abolish the production of the truncated transcripts in vivo also exhibit reduced promoter activity in vitro. The appearance of high levels of truncated transcripts raise the interesting possibility that-similar to c-myc, c-myb, and c-fos--basal HIV-1 expression is regulated by transcription elongation.
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PMID:Sequences within the R region of the long terminal repeat activate basal transcription from the HIV-1 promoter. 145 Jun 62

Previously we have demonstrated the existence of stable transcripts from the noncoding strand of a rearranged c-myc gene in murine plasmacytomas in which the oncogene has translocated to an immunoglobulin constant-region gene element (M. Dean, R. B. Kent, and G. E. Sonenshein, Nature [London] 305:443-446, 1983). The resulting RNAs are chimeric, containing c-myc antisense and immunoglobulin sense sequences. A normal unrearranged murine c-myc gene is transcribed in the antisense orientation throughout much of the gene; however, stable transcripts have not been detected. In this study, using Northern (RNA) blot, S1 nuclease, and primer extension analyses, we have mapped the 5' end of the stable chimeric transcripts to a site 175 bp from the start of exon 3, within intron 2 of the c-myc gene. In vitro transcription assays with constructs containing this site and 400 bp upstream, in the antisense orientation, and nuclear extracts from plasmacytoma cells, as well as a number of cell lines with normal unrearranged c-myc genes, indicated that this promoter was functional. This finding was confirmed in transient transfection assays using the antisense promoter linked to the chloramphenicol acetyltransferase reporter gene. These results suggest that a normal promoter of antisense transcription is used following c-myc gene translocation.
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PMID:An antisense promoter of the murine c-myc gene is localized within intron 2. 154 13

To identify the regions in the chicken c-myc promoter that are necessary for the binding of a nuclear trans-acting factor CTCF--the potential oncogene activator--we used a synthetic analog of the natural binding site that contains three correctly spaced CCCTC-repeats that are known to be involved in CTCF-binding. Gel retardation experiments failed to detect any CTCF-binding activity with this synthetic site. We conclude that GC-transversions made in the regions presumed to be invalid, do in fact interfere with the protein binding. The secondary structure analysis with S1-nuclease shows the presence of an unusual DNA conformation of the CTCF-binding site in the supercoiled plasmids, that can not be detected with the artificial construction. The precise mapping of S1 nuclease cleavage reveals several hypersensitive sites in the CCCTC-zone. Thus, an altered secondary structure may be functionally important for the protein recognition in vivo.
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PMID:[Regulatory protein factor CTCF interacts with a segment of the chicken c-myc oncogene promotor, capable of changing to a noncanonical conformation]. 179 97

In Burkitt's lymphoma cells the c-myc gene locus is consistently fused to the constant region of one of the immunoglobulin genes by chromosomal translocation. The translocated c-myc gene is transcriptionally activated and preferentially transcribed from the P1 promoter whenever the exon-intron structure of c-myc remains intact. In order to define elements involved in this promoter shift we have cloned the translocated c-myc allele from Burkitt's lymphoma cell line BL60, which is characterized by several point mutations. The mutated c-myc allele of BL60 was stably introduced into baby hamster kidney and Burkitt's lymphoma cells. S1 nuclease and RNAase protection mapping experiments demonstrated that the mutated c-myc allele was expressed at a low level and with a normal promoter usage (P2 greater than P1) in Burkitt's lymphoma and baby hamster kidney cells. Furthermore, we have studied the expression of a construct consisting of the mutated c-myc allele, part of the bvr1 (Burkitt's variant rearranging region 1) locus, the human immunoglobulin kappa constant region, and the kappa intron enhancer after stable transfection into Burkitt's lymphoma cells. Although c-myc expression was about fivefold increased, the transcripts still initiated predominantly at promoter P2. This indicates that 5 kb of the constant kappa light-chain locus including the kappa intron enhancer is not sufficient to induce the Burkitt's lymphoma-specific promoter shift.
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PMID:The intron enhancer of the immunoglobulin kappa gene activates c-myc but does not induce the Burkitt-specific promoter shift. 194 9

The c-myc gene is rapidly induced in quiescent Balb/c-3T3 cells in response to the platelet-derived growth factor (PDGF). In order to study the mechanisms by which growth factors regulate induction of c-myc, we have attempted to identify growth factor-responsive elements in the murine c-myc locus. Various fragments of the c-myc gene linked to a bacterial CAT reporter gene were stably transfected into Balb/c-3T3 cells. A construct which includes the P1 promoter and 424 bp of upstream sequences shows a 3-5 fold induction of CAT RNA expression in response to sis/PDGF. S1 nuclease mapping experiments demonstrate that this mRNA initiates from the myc P1 promoter. Nuclear runoff transcription experiments performed with this myc/CAT construct show that this induction occurs at the transcriptional level. Deletion analysis led to the identification of an 81 bp segment in the first exon between the c-myc P1 and P2 promoters which is necessary to confer growth factor responsiveness in this construct.
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PMID:Identification of a PDGF-responsive element in the murine c-myc gene. 225 Sep 9

Transcription of the c-myc gene is initiated mainly from two promoters, P1 and P2. By S1 nuclease analysis we found that there is 8 times more P2- than P1-initiated RNA in total RNA from HL60 cells. The half-lives of P1- and P2-initiated transcripts are 26 and 18 min, respectively, so the difference in the relative abundance of the mRNAs is not due to differences in their stabilities. The relative rates of transcription from the P1 and P2 promoters, estimated by in vitro nuclear run-on analysis, were found to differ by about 10-fold, sufficient to account for the difference in the steady-state levels of the two mRNAs. The abundance of c-myc mRNA changes dramatically during differentiation of HL60 cells. Dimethyl sulphoxide causes a very rapid reduction in total c-myc mRNA, while with phorbol ester a transient increase occurs followed by a more gradual decline. At no time during these dramatic alterations were significant changes detected in the relative abundance of P1- and P2-initiated mRNAs, or in their stabilities.
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PMID:Regulation of the relative abundances of c-myc mRNAs in human promyelocytic HL60 cells. 229 63

During post-natal cerebellar development the steady-state levels of c-myc transcripts exhibit characteristic changes. As determined by the S1 nuclease protection assay the level of c-myc transcript, which is very high in the late embryonic cerebellum, decreased to low levels shortly after birth. One week later there is a second period of c-myc mRNA accumulation followed by a marked decline to finally reach the low adult value. The second peak of high c-myc mRNA level correlates well with the proliferation of granule cell precursors, and it is characterized by a marked change in the ratio of the two types of transcripts started at the known c-myc promoters 1 and 2. This indicates a change in the cell population involved in the transcription of the c-myc gene. In situ hybridization shows transiently elevated c-myc mRNA levels in neurons of the cerebellar cortex. At post-natal days 3 and 10 (P3 and P10) c-myc transcripts are detectable in the superficial external granular layer composed primarily of mitotically active (neural precursor) cells. Purkinje cell somata show cytoplasmic label at P10. These large postmitotic neurons undergo rapid differentiation at this developmental stage. In the adult cerebellum the low c-myc mRNA level is apparently due to Purkinje cells with barely detectable amounts of c-myc transcripts. The vast majority of mature cerebellar neurons, the internal granule cells, have no specific hybridization signal for c-myc. We conclude that neurons in vivo can accumulate c-myc messenger during proliferation and/or differentiation, perhaps as a cellular response to an external signal.
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PMID:Proto-oncogene c-myc is expressed in cerebellar neurons at different developmental stages. 242 14

We have produced transgenic mice carrying an H-2K/human c-myc fusion gene. In this construct, the human c-myc proto-oncogene expression is driven by the 5' flanking sequences (including promoter) of the class I H-2Kb gene, which have previously been shown to direct the expression of a marker gene, the human growth hormone (hGH), in most tissues of H-2K/hGH transgenic mice. Comparative analysis, by S1 nuclease mapping, of the H-2K and human c-myc gene expression in different organs of the H-2K/myc mice shows that exogenous c-myc and endogenous H-2K expression is found in most organs examined. However, the liver is a notable exception, for here c-myc expression is very weak. The exogenous c-myc expression is maximal in lymphoid organs of all H-2K/myc transgenic strains. One strain, H-2K/myc 27, hereditarily develops a lymphoproliferative syndrome which eventually leads to death. The H-2K/myc 27 lymphoid tissues are profoundly abnormal: pre-B cells as well as mature B cells are underrepresented in the bone marrow but the thymus as well as lymph nodes are largely infiltrated by B cells. Moreover, in the thymus, the proportions of the different thymic cell populations are altered. However, in the other H-2K/myc transgenic strains, even in those expressing a comparable or even higher level of myc, no pathology has been observed over a period of 20 months. Our results, therefore, demonstrate that constitutive enforced c-myc expression might disturb lymphocyte development, but does not directly lead to malignancy.
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PMID:Lymphoproliferative syndrome associated with c-myc expression driven by a class I gene promoter in transgenic mice. 265 14

Accumulation of unusually high amounts of larger-than-normal c-myc mRNAs occurs in two mouse plasmacytomas, TEPC 1165 and TEPC 2027. Southern blot and DNA sequence analyses showed that both tumors have undergone translocations of immunoglobulin heavy chain loci to positions 5' of the c-myc gene promotors resulting in removal of DNA sequences encoding a negative transcriptional regulatory element. In contrast to other mouse plasmacytomas, TEPC 1165 and TEPC 2027 rearranged myc genes show increased transcription, partially explaining their abundance of myc RNA. Similar to other mouse plasmacytomas, the abundance of myc RNA in TEPC 1165 and TEPC 2027 is also influenced by increased stability of structurally atypical myc RNAs. Two myc mRNAs are found in TEPC 2027, a 2.4 kb species including all 3 myc exons and a 4.0 kb species with the 3 exons plus the first intron. The two major myc mRNAs in TEPC 1165, 3.0 and 3.9 kb species, also include all three myc exons plus portions of the first intron. S1 nuclease protection analyses show that the 5' initiation and 3' untranslated (UT) regions of the unusual TEPC 1165 RNAs are normal showing that the size differences arise solely from inclusion of first intron sequences in the large myc RNAs. DNA sequence analysis showed that the presence of first intron sequences in the large myc RNAs is due to mutations affecting the splice donor region at the 3' end of exon 1 in both tumors. SDS-PAGE analysis of immunoprecipitated TEPC 1165 and TEPC 2027 myc proteins showed them to be of normal electrophoretic mobility but no more abundant than in a pre-B cell line 18-81 that contains at least 10 fold less myc RNA. The 4.0 kb myc mRNA of TEPC 2027 is atypically stable while the 2.4 kb myc mRNA undergoes normal rapid turnover within the same cell, demonstrating that the presence of first intron sequences in the large myc RNA stabilizes it despite the presence of 3' UT and putative exon 1 destabilizing sequences. These results show that myc intron 1 sequences can counteract the effect of 3' UT region destabilizing sequences in myc RNA and suggest that the increased myc RNA stability noted in TEPC 1165 and TEPC 2027 is largely due to the presence of the intron 1 sequences.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Altered myc gene transcription and intron-induced stabilization of myc RNAs in two mouse plasmacytomas. 265 76


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