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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper further characterizes the estrogen-binding protein we have described in the cytosol of the yeast Saccharomyces cerevisiae. [3H]Estradiol was used as the radioprobe, and specific binding of cytosol fractions was measured by chromatography on Sephadex minicolumns. Other 3H-steroids did not exhibit specific binding. [3H]Estradiol binding was destroyed by treatment with trypsin, but not RNase, DNase, or phospholipase; N-ethylmaleimide substantially decreased the binding. The yeast did not metabolize estradiol added to the medium, and extraction and chromatography of the bound moiety showed it to be unmetabolized estradiol. Scatchard analysis of cytosol from both a and alpha mating types as well as the a/alpha diploid cell revealed similar binding properties: an apparent dissociation constant or Kd(25 degrees) for [3H]estradiol of 1.6-1.8 nM and a maximal binding capacity or Nmax of approximately 2000-2800 fmol/mg of cytosol protein. Gel exclusion chromatography on Sephacryl S-200 and high performance liquid chromatography suggested a Stokes radius of approximately 30 A. Sucrose gradient centrifugation showed a sedimentation coefficient of approximately 5 S, and the complex did not exhibit ionic dependent aggregation. The estrogen binder in S. cerevisiae differed in its steroidal specificities from classical mammalian estrogen receptors in rat uterus. 17 beta-Estradiol was the best competitor, 17 alpha-estradiol had about 5% the activity, and diethylstilbestrol exhibited negligible binding affinity as did tamoxifen, nafoxidine, and the zearalenones. In summary, a high affinity, stereospecific, steroid-selective binding protein has been demonstrated in the cytosol of the simple yeast S. cerevisiae. We speculate that this molecule may represent a primitive hormone receptor system, possibly for an estrogen-like message molecule.
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PMID:Characterization of an estrogen-binding protein in the yeast Saccharomyces cerevisiae. 636 45

RNase A treatment of HeLa cell nuclei causes a time- and concentration-dependent release of dexamethasone-receptor complexes. If nuclei are incubated in the absence of enzyme, only 60% of RNase-releasable complexes can be detected. Sucrose density gradient analysis of nuclear extracts shows that receptor complexes released by RNase treatment sediment at 3.6 S, whereas complexes obtained from untreated nuclei sediment between 7 and 3.6 S. Our results show that a fraction of dexamethasone-receptor complexes retained by HeLa cell nuclei is located in binding sites involving RNA.
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PMID:RNA-containing nuclear binding sites for glucocorticoid-receptor complexes. 647 89

The 7-8 S form of the [3H]dexamethasone (9 alpha-fluoro-11 beta,17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3, 20-dione) receptor from rat liver cytosol can be converted to the 3-4 S form by RNase treatment or high salt, suggesting a salt-sensitive association between the receptor protein and RNA. In DNA-cellulose column assays, the gradient-purified 3-4 S form bound DNA more efficiently than the 7-8 S form, though the 7-8 S form was also capable of binding to DNA-cellulose to a significant extent. Activated 7-8 S dexamethasone receptor could be released from its association with soluble DNA by treatment with DNase I. Sucrose gradient analysis showed that the released receptor sedimented as the 7-8 S form and was sensitive to RNase treatment, which induced a conversion to the 3-4 S form. Activated RNase-generated 3-4 S receptor again displayed a higher degree of binding to soluble DNA and was recovered in the 3-4 S form following DNase extraction. The fact that the 3-4 S form bound immobilized or soluble DNA more efficiently suggests that the associated RNA of the 7-8 S form interferes directly or indirectly with the receptor association with DNA. The observation that the receptor binds to DNA in its 7-8 S form suggests that the receptor complex is capable of binding RNA and DNA concurrently.
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PMID:Binding of the rat liver 7-8 S dexamethasone receptor to deoxyribonucleic acid. 671 44

The in vitro nuclear binding of rat uterine estrogen-receptor complexes has been studied. Heating cytosol from mature rat uterus at 25 C for various times in the presence of 0.15 M KCl resulted in a transient increase in nuclear binding activity, followed by irreversible loss of this activity. The molecular state of these complexes heated at 25 C in the presence of 0.15 M KCl was determined using Sephadex G-200 chromatography and sucrose density centrifugation at high ionic strength (0.4 M KCl). Gel filtration resulted in steroid-binding activity in the void volume. Sucrose density gradient analysis revealed a broad peak, ranging from approximately 5-20S. When cytosol was heated at 25 C in the presence of 10 mM molybdate to block the temperature-induced activation of receptor, nuclear binding ability was easily recovered by dialysis, while heating already activated estrogen receptor in the presence of 0.15 M KCl and 10 mM molybdate caused irreversible loss of nuclear binding ability. When cytosols prepared from immature rats (19-23 days old) were heated at 25 C in the presence of 0.15 M KCl, only a minimum loss of nuclear binding ability was shown. The radioactive peak in a high salt sucrose density gradient appeared almost exclusively in the 5S region. However, the addition of receptor-free mature uterine cytosol to estrogen-receptor complexes from immature rat uterus caused a marked loss of nuclear binding utility, with a resultant receptor aggregation, whereas rat liver cytosol had no effect on this reaction. Furthermore, heating liver glucocorticoid receptor did not cause a loss of nuclear binding ability even in the presence of receptor-free adult rat uterine cytosol. These observations suggest that there is a factor(s) in rat uterus which recognizes only activated estrogen receptor and induces receptor aggregation and a rapid loss of the nuclear binding ability of receptor in a KCl concentration- and temperature-dependent manner. Preliminary characterization indicates that this factor is macromolecular in nature and resistant to RNase and trypsin treatment, but labile at 100 C.
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PMID:A modulator which converts activated estrogen receptor to a biologically inactive aggregated form. 679 27

Antibodies to the RNase-sensitive RNP and to the RNase-resistant Sm nuclear antigens were used to affinity purify these antigens from a saline extract of rabbit thymus acetone powder. Determination of the protein subunits recovered by either glycine-HCl, pH 2.8, or 2.5 M MgCl2 elution on gradient sodium dodecyl sulfate-polyacrylamide electrophoresis containing mercaptoethanol revealed that RNP was composed of five proteins with mol. wts from 10,000 to 15,000 whereas Sm contained the same or similar five chains plus six additional subunits with mol. wts from 21,000 to 42,000. RNase treatment of the thymus extract increased the recovery in Sm of the same bands compared to untreated extract. Thus, RNP and Sm appear to have different numbers of protein components and RNP may be a subset of Sm. Sucrose gradient centrifugation of the 125I-labeled, pH 2.8 eluted antigens gave peaks of 3 and 6S for RNP and Sm, respectively. Sucrose gradient centrifugation of the crude untreated thymus extract followed by quantitative single radial immunodiffusion analysis of each fraction produced a broad peak from 16S to the top of the gradient while pretreatment of the extract with RNase resulted in a discrete 6S peak. These results indicate that in rabbit thymus acetone powder native RNP and Sm exist as larger polydisperse complexes with additional material including RNA and that after acid elution or RNase treatment the antigens are found in a smaller monodisperse form.
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PMID:Characterization of RNP and Sm ribonucleoprotein nuclear antigens. 681 Jan 1

A monoclonal antibody from clone T7 raised against total nuclear proteins from the Kc cell line of Drosophila melanogaster (Saumweber, H. Symmons. P. Kabisch, R. Will, H & Bonhoeffer, F, Chromosoma 80 (1980) 253) [1] showed positive immunofluorescent staining on interphase nuclei of HeLa and PTK2 cells. When this antibody was allowed to react with several nuclear protein fractions isolated from HeLa S3 cells, three polypeptides of molecular weights (MW) 44 000, 63 000 and 70 000 were identified as the corresponding antigens, all components of hnRNA containing ribonucleoprotein particles. Sucrose gradient fractionation of such particles after mild RNase treatment and subsequent analysis of the proteins by the immunoblotting method revealed that the 44 000 MW antigen was an integral part of the ribonuclease-resistant complex. The results support the view that hnRNA molecules are associated with certain proteins conserved during evolution which may play structural roles in the ribonucleoprotein organization.
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PMID:Cross-reaction of hnRNP-proteins of HeLa cells with nuclear proteins of Drosophila melanogaster demonstrated by a monoclonal antibody. 681 36

The interactions between proteins and solvent components have been investigated for the sucrose/water system. Thermodynamic and kinetic measurements of the thermal unfolding of alpha-chymotrypsin, chymotrypsinogen, and ribonuclease were performed as a function of sucrose concentration. The alteration in protein-solvent interactions in the presence of sucrose was also studied by density measurements and analyzed by multicomponent thermodynamic theory. Sucrose does not induce a conformational change in three proteins studied, although it does induce a small change in the circular dichroism spectrum of ribonuclease. The enthalpy of thermal unfolding shows little dependence on the concentration of sucrose, while the apparent activation energy of the unfolding process is increased by the addition of sucrose. The results from the protein-solvent interaction study indicate that sucrose is preferentially excluded from the protein domain, increasing the free energy of the system. Thermodynamically this leads to protein stabilization since the unfolded state of the protein becomes thermodynamically even less favorable in the presence of sucrose. The exclusion of sucrose from the protein domain seems to be related to the higher cohesive force of the sucrose water solvent system since all the experimental observations can be correlated with the effect of sucrose on the surface tension of water.
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PMID:The stabilization of proteins by sucrose. 725 92

The nuclear distribution of Drosophila DNA topoisomerase II was determined by immunoblot analysis after nuclease digestion and cell fractionation. About 60% of DNA topoisomerase II could be removed from nuclei by RNase A, about 70% by DNase I, and about 90% by incubation with both enzymes together or with micrococcal nuclease. Nuclease treatment of nuclei did not affect the distribution of lamins Dm1 and Dm2 or other nuclear proteins similarly. Nuclease-mediated solubilization of DNA topoisomerase II from Drosophila nuclei was also dependent on NaCl concentration. Solubilization was not efficient below 100 mM NaCl. Sucrose velocity gradient ultracentrifugation demonstrated that DNA topoisomerase II solubilized from nuclei by either RNase A or DNase I migrated at about 9 S, as expected for the homodimer. Results of chemical crosslinking supported this observation. We conclude that DNA topoisomerase II has both RNA- and DNA-dependent anchorages in Drosophila embryo nuclei.
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PMID:Nuclear distribution of Drosophila DNA topoisomerase II is sensitive to both RNase and DNase. 761 83

Of the 15 nicotinic ACh receptor genes identified in vertebrates, only four (alpha 1, beta 1, gamma, and delta) have been shown to be expressed in embryonic skeletal muscle at early times. In mammalian muscle a fifth gene (epsilon) replaces the gamma gene in expression at later times. The remaining 10 nicotinic receptor genes identified to date (alpha 2-alpha 8, beta 2-beta 4) are expressed in the nervous system and are considered neuronal genes. Using RNase protection assays, we show here that four of the neuronal-type genes (alpha 4, alpha 5, alpha 7, and beta 4) are expressed in developing chick skeletal muscle. Two of them (alpha 4 and alpha 7) decline substantially in transcript abundance between embryonic days 11 and 17, as does alpha 1, while the other two (alpha 5 and beta 4) show only moderate decreases over the same time period. At embryonic day 8, alpha 7 transcripts are nearly 20% as abundant as alpha 1 transcripts. In situ hybridizations confirm the presence of alpha 7 transcripts in muscle cells both in cell culture and in embryonic tissue. No evidence was found for expression of the alpha 2, alpha 3, alpha 8, or beta 3 genes in muscle. Immunoprecipitations and immunoblot analysis using subunit-specific monoclonal antibodies reveal alpha 7 protein in muscle, and the amount of protein rises and declines with the amount of alpha 7 mRNA during development. Sucrose gradient analysis demonstrates that the alpha 7 protein is present in muscle as a species of 10S, the size expected for a nicotinic receptor. The alpha 7 species in muscle binds alpha-bungarotoxin but does not contain alpha 1 subunits, indicating that the two kinds of alpha-type gene products segregate during assembly. The results suggest that neuronal AChRs may play a role in early muscle development.
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PMID:Expression of neuronal acetylcholine receptor genes in vertebrate skeletal muscle during development. 786 4

The polyadenylate tail of eukaryotic mRNAs is thought to influence various metabolic phenomena including mRNA stability, translation initiation, and nucleo-cytoplasmic transport. We have analyzed the fate of mRNAs following inactivation of poly(A) polymerase in Saccharomyces cerevisiae containing a temperature-sensitive, lethal mutation (pap1-1) in the gene for poly(A) polymerase (PAP1). Inactivation of poly(A) polymerase (Pap1) by shifting cells to the nonpermissive temperature resulted in the loss of at least 80% of measurable poly(A) within 60 min. Northern blot analysis revealed the disappearance of some mRNAs (CYH2 and HIS4) consistent with a role for poly(A) tails in mRNA stability. However, other mRNAs (TCM1, PAB1, ACT1, and HTB2) accumulate as poly(A)-deficient (A < approximately 25) transcripts as defined by an inability to bind oligo(dT)-cellulose. Sucrose density gradient analysis of polyribosomes revealed a twofold reduction in the amount of each size class of polyribosomes in shifted cells and a commensurate increase in free ribosomes. However, poly(A)-deficient mRNAs in shifted cells remain associated with the same size polyribosomes as poly(A)+ mRNAs in unshifted cells, indicating normal initiation of translation. RNase mapping of transcripts from pap1-1 cells revealed PAB1 mRNA to be poly(A)- whereas TCM1 exists as equal amounts of poly(A)- and poly(A)+ mRNA 60 min after shift. Interestingly, both of these classes of TCM1 mRNA appear in similar amounts in each polyribosome fraction indicating that ribosomes may not distinguish between them. These findings suggest that under conditions of excess translational capacity, poly(A)- and poly(A)+ mRNAs may initiate translation with comparable efficiencies.
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PMID:Efficient translation of poly(A)-deficient mRNAs in Saccharomyces cerevisiae. 795 21


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