Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have expressed in the yeast Saccharomyces cerevisiae a full-length poliovirus cDNA clone under the control of the GAL10 promoter to better characterize the effect of poliovirus on host cell metabolism. We find that yeast cells are unable to translate poliovirus RNA in vivo and that this inhibition is mediated through the 5' untranslated region of the viral RNA. The in vivo inhibition of translation of poliovirus RNA and P2CAT RNA (which contains the 5' untranslated region fused upstream of the bacterial chloramphenicol transferase gene) can be mimicked in vitro in yeast translation lysates. In fact, a trans-acting inhibitor present in yeast lysates can inhibit translation of either poliovirus or P2CAT RNA in HeLa cell translation lysates. In contrast, when the inhibitor is added to translations programmed with chloramphenicol acetyltransferase RNA, yeast prepro-alpha-factor RNA, or an RNA containing the internal ribosome entry site of encephalomyocarditis virus, no inhibition is seen. The inhibitory activity has been partially purified by DEAE-Sephacel chromatography. The partially purified inhibitor is heat stable, escapes phenol extraction, is resistant to proteinase K and DNase I treatment, and is sensitive to RNase A digestion, suggesting that the inhibitor is an RNA. In an in vitro translation assay, the inhibitory activity can be overcome by increasing the concentration of HeLa cell lysate but not P2CAT RNA, suggesting that the inhibitor interacts (directly or indirectly) with one or more components of the HeLa cell translational machinery rather than with the viral RNA.
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PMID:Yeast cells are incapable of translating RNAs containing the poliovirus 5' untranslated region: evidence for a translational inhibitor. 130 48

Nuclear run-on transcription assays allow the study of those factors that regulate the expression of a given gene. Because previously published protocols may have been presented in abbreviated form or not developed for nuclei derived from specific tissues, we have established a comprehensive protocol for those who wish to measure transcription of the gonadotropin subunit genes (alpha, LH-beta, FSH-beta) in the anterior pituitary of the rat. The protocol also allows the determination of simultaneously existing cytoplasmic subunit mRNA levels within the same cells. An especially critical step in nuclear run-on transcription assays is the isolation of newly formed labeled mRNA transcripts from the DNA template. In previously published protocols such isolation has been accomplished through rather tedious, multistep methods. To simplify this isolation process, we have evaluated the use of the commercially available product RNAzol. Nascent mRNA transcripts are attached to chromatin and the DNA template; proteinase K and DNase I are typically utilized to disrupt this attachment, thereby facilitating subsequent extraction. We have found that strict adherence to proteinase K and DNase I digestion prior to RNA extraction with RNAzol, followed by two ethanol/NH4OAc precipitations, will enable one to successfully isolate nascent mRNA transcripts free of unincorporated nucleotides. In summary, we present a comprehensive protocol for determining transcription rates and cytoplasmic mRNA levels for the gonadotropin subunit genes in the anterior pituitary of the rat. RNAzol was found to simplify the extraction of newly formed transcripts and should prove applicable with transcription assays utilizing nuclei from a variety of tissue.
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PMID:Determination of coexisting nuclear transcription rates and cytoplasmic mRNA levels for gonadotropin subunit genes in rat anterior pituitary. 137 79

The interferon induced double-stranded-RNA-dependent eIF-2 alpha kinase has an established role in mediating part of interferons anti-viral effects. Several studies have suggested that it may have additional functions in cells not infected with virus. The mechanism of activation of the kinase and the consequences of its activity in uninfected cells remain to be determined. Our previous results have indicated that the activation (phosphorylation) of this kinase may be an important regulatory signal to the arrest of growth of mouse 3T3-F442A fibroblasts and their subsequent differentiation to adipocytes. We have found that the phosphorylation of the kinase occurred in vivo in the absence of viral infection and in vitro without the addition of dsRNA. We demonstrate here that total cytoplasmic RNA from 3T3-F442A cells contains a regulatory RNA(s) capable of activating dsRNA-dependent eIF-2 alpha kinase. Fractionation of the cytoplasmic RNA by oligo(dT)-cellulose indicated that the regulatory RNA eluted with the poly(A)-rich RNA fraction. It bound tightly to the dsRNA-dependent eIF-2 alpha kinase and was immune-precipitated with its antibodies as a complex of regulatory RNA and dsRNA-dependent eIF-2 alpha kinase. The regulatory RNA activity was further purified by phenol extraction of immune precipitates containing this complex. These findings indicated that the regulatory RNA forms a specific complex with the dsRNA-dependent eIF-2 alpha kinase. The activity of the regulatory RNA was sensitive to the dsRNA-specific RNase VI but not to proteinase K, DNase I or ssRNA-specific RNase T1. The activation of the dsRNA-dependent eIF-2 alpha kinase by regulatory RNA was prevented by addition of a high concentration of poly(I).poly(C). The regulatory RNA was also shown to activate partially purified dsRNA-dependent eIF-2 alpha kinase prepared from rabbit reticulocyte lysates and to inhibit protein synthesis in reticulocyte lysates. Our findings, that cellular RNAs can specifically activate the dsRNA-dependent eIF-2 alpha kinase, are consistent with a physiological role for the dsRNA-dependent eIF-2 alpha kinase and interferon during cell growth and differentiation. The relationship of the regulatory RNA activity to growth and differentiation of 3T3-F442A cells is discussed.
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PMID:Activation of the double-stranded RNA-dependent eIF-2 alpha kinase by cellular RNA from 3T3-F442A cells. 170 58

Immunohistochemical studies with antiserum against the protamines of the toad, Bufo japonicus, revealed that the sperm nucleus loses protamines within 5 min after entry into the egg. Likewise, lysolecithin-permeabilized sperm incubated with the egg extract lose the protamines within 1 min, accompanied by nuclear decondensation. The activities that induce both protamine removal and decondensation in sperm nuclei were found in extracts from growing and mature oocytes and pregastrula embryos, but not in postneurula embryos or adult tissues. SDS-PAGE analyses revealed that the egg extract removed not only protamines from the Bufo sperm, but also selectively the sperm-specific basic proteins from sperm nuclei of Xenopus laevis. The protamine-removing activity (PRA) was partially purified from egg extracts as negatively charged macromolecules by anion-exchange chromatography and gel filtration. The PRA was heat-stable (100 degrees C, 10 min) and sensitive to proteinase K, but not to RNase A and DNase I. Immunoblot analysis of the supernatant after incubation of Bufo sperm in the fraction with the PRA revealed that protamines derived from sperm nuclei were associated with a major protein of the fraction. This protein exhibited mobilities of 140 and 36 kDa on native- and SDS-PAGE, respectively, with the isoelectric points in the range 4.2 to 4.5 and possessed an amino acid composition quite similar to that reported for Xenopus nucleoplasmin. We propose that in fertilized eggs the protamines are removed from sperm nuclei by nucleoplasmin by binding to but not by enzymatic degradation of the protamine.
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PMID:Characterization of the ooplasmic factor inducing decondensation of and protamine removal from toad sperm nuclei: involvement of nucleoplasmin. 193 66

The rat hepatic S14 gene is regulated by L-triiodothyronine (T3) and codes for a cytosolic protein (pI 4.9 and Mr 17,010) that is believed to be involved in lipogenesis. Recent studies have identified at least five DNase I hypersensitive sites (HS 1-5) in hepatic chromatin flanking the 5' region of the gene. The HS-1 site is situated immediately adjacent to the transcription initiation site. We have isolated a DNA fragment (USS-1) which contains a portion of the HS-1 site to examine the binding of nuclear proteins to S14 DNA. DNase I footprinting studies demonstrated that material extracted from hepatic nuclei with 0.42 M NaCl contained proteinase K-sensitive factors (presumed to be proteins), which bind to USS-1 DNA between positions -63 and -48 (PS-1) relative to the transcription initiation site. Examination of the binding activity with a synthetic oligonucleotide identical to the protected sequence indicated the formation of at least three protein-DNA complexes. The DNA binding activity of the PS-1 binding protein or proteins correlated with the T3 regulated expression of mRNA-S14. Although the nucleotide sequence of PS-1 closely resembles the binding site for the CCAAT transcription factor (CTF/NF-1), competition studies attempting to displace protein binding from the PS-1 sequence with DNA fragments containing the CTF/NF-1 binding motif were unsuccessful. In vitro transcriptional assay studies suggested that the DNA fragment (-441 to -2) containing the PS-1 site promotes the transcription of the S14 gene in an orientation fashion. The in vitro transcriptional activity of the S14 DNA containing the PS-1 sequence was significantly higher in hepatonuclear extracts from hyperthyroid compared with euthyroid or hypothyroid animals. In summary, our findings indicate that the DNA binding activity of proteins which bind to PS-1 site is influenced by the thyroid status of the animal.
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PMID:Binding of nuclear proteins to the rat liver S14 gene is influenced by thyroid state. 234 5

Z chromatin-chromium (Cr) complex, prepared from mouse liver chromatin and CrCl3, showed a significantly enhanced template activity for in vitro RNA synthesis. Digestion experiments with this complex using micrococcal nuclease and DNase I suggested that Cr(III) preferentially binds to linker regions rather than core regions of chromatin. Further, it was found that Cr(III) binds to DNA and nonhistone proteins (NHP), but hardly to histones. Moreover, the template activity of an NHP-Cr complex, when added to a DNA-histones complex, was inhibited remarkably. The template activity of the chromatin-Cr complex was not significantly altered by proteinase K digestion. Furthermore, experiments using rifampicin and [gamma-32P]guanosine 5'-triphosphate (GTP) demonstrated an increase in the number of initiation sites in the chromatin-Cr complex. These results suggest that, in this in vitro system, Cr(III) preferentially binds to DNA in chromatin and causes an increase in the number of initiation sites, thus enhancing RNA synthesis.
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PMID:Enhancement of in vitro ribonucleic acid synthesis on chromium(III)-bound chromatin. 242 31

A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.
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PMID:Reverse differential staining of sister chromatids induced by Hoechst plus black light and endonuclease. 257 33

The mechanism of chromosome banding induced by restriction endonucleases was analyzed by measuring the amount of radioactivity extracted from [14C]thymidine-labeled chromosomes digested first with restriction enzymes and subsequently with proteinase K and DNase I. Restriction enzymes with a high frequency of recognition sites in the DNA produced a large number short DNA fragments, which were extracted from chromosomes during incubation with the enzyme. This loss of DNA resulted in decreased chromosomal staining, which did not occur in regions resistant to restriction enzyme digestion and thus led to banding. Subsequent digestion of chromosomes with proteinase K produced a further loss of DNA, which probably corresponded to long fragments retained in the chromosome by the proteins of fixed chromatin. Restriction enzymes induce chromatin digestion and banding in G1 and metaphase chromosomes, and they induce digestion and the appearance of chromocenters in interphase nuclei. This suggests that the spatial organization and folding of the chromatin fibril plays little or no role in the mechanism of chromosome banding. It was confirmed that the pattern of chromosome banding induced by AluI, MboI, HaeIII, DdeI, RsaI, and HinfI is characteristic for each endonuclease. Moreover, several restriction banding polymorphisms that were not found by conventional C-banding were detected, indicating that there may be a range of variability in the frequency and distribution of restriction sites in homologous chromosome regions.
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PMID:The mechanism and pattern of banding induced by restriction endonucleases in human chromosomes. 298 44

We have previously shown that exposure of responding cells to vitamin A leads to profound modifications of chromatin structure as revealed by an increased susceptibility to DNase I digestion, modified patterns of histone acetylation, and impaired synthesis of a nonhistone chromosomal protein (Ferrari, N., and Vidali, G. (1985) Eur. J. Biochem. 151, 305-310). The present results show that these effects are most probably due to the direct interaction between retinol and chromatin, and analysis of mononucleosomes and higher oligomers obtained from retinol-treated cells shows that retinol is indeed tightly bound to chromatin. Enzymatic digestions of vitamin A containing nucleosomes with proteinase K, phospholipase C, and phospholipase A2 support a model where the final binding of retinol to chromatin is mediated by a lipoprotein: the recognition of the binding sites on DNA being dictated by the proteic component while the hydrophobic retinol is solubilized in the fatty acid moiety.
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PMID:In vivo binding of retinol to chromatin. The binding is mediated by a lipoprotein. 333 5

DNA topology in histone- and protamine-depleted nuclei (nucleoids) from somatic cells, sperm, and spermatogenic cells was studied to determine if the superhelical configuration of DNA looped domains is altered during spermatogenesis. The expansion and contraction of nucleoid DNA was measured with a fluorescence microscope following exposure of nucleoids to different concentrations of ethidium bromide (EB). Nucleoids from Xenopus laevis erythrocytes, primary spermatocytes, and round spermatids, and from Rana catesbeiana sperm all exhibited a biphasic change (condensed-relaxed-condensed) in size as a function of exposure to increasing concentrations (0.5-100 micrograms/ml) of EB, indicating that they contain negatively supercoiled DNA. In contrast, DNA in sperm nucleoids from Xenopus laevis and Bufo fowleri was relaxed and expanded at low (0.5-6 micrograms/ml) EB concentrations, but became gradually condensed as the EB concentration was increased (6-100 micrograms/ml). Nucleoids prepared from all cell types retained the general shape of the nucleus regardless of the superhelical configuration of the nucleoid DNA. Sperm nucleoid DNA condensed by 100 micrograms/ml EB was relaxed by exposure to UV light, DNase I, proteinase K, or 4 M urea, but not by RNase A or 10 mM dithiothreitol. These results demonstrate that the DNA in sperm nucleoids is constrained in domains of supercoiling by nonbasic nuclear proteins. Negatively supercoiled DNA is present in nucleoids from cells with a full complement of histones, including Rana sperm, but not in nucleoids from Xenopus and Bufo sperm in which histones are replaced by "intermediate-type" protamines. Histone replacement in these species, therefore, is accompanied by unfolding of nucleosomal DNA and active removal of the negative supercoils. Results presented also suggest an important role for the nonbasic nuclear proteins of sperm in the morphogenesis of the nucleus and the arrangement of DNA.
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PMID:Changes in DNA topology during spermatogenesis. 349 Mar 60


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