Purification of Neurospora crassa endonuclease-resistant DNA–RNA hybrid

1970 ◽  
Vol 48 (4) ◽  
pp. 501-507 ◽  
Author(s):  
M. J. Fraser ◽  
E. Z. Rabin ◽  
G. Allen
Keyword(s):  
Neurospora Crassa ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Calf Thymus Dna ◽  
Degraded Dna ◽  
Sucrose Density Gradient Centrifugation ◽  
Calf Thymus

Denatured calf thymus DNA has been transcribed in vitro with RNA polymerase of Micrococcus lysodeikticus. The DNA–RNA hybrid which formed in this system was found to be highly resistant to a purified Neurospora crassa endonuclease, while the unhybridized DNA and RNA were rapidly degraded. DNA–RNA hybrid with a purity of 62–75% was isolated in one step from the digest in 69–86% yield by chromatography on methylated albumin – kieselguhr. Further purification was obtained by density gradient centrifugation in Cs2SO4 solution. The purified hybrid had a buoyant density of 1.49 g cm−3 in Cs2SO4, intermediate between that of mouse tRNA marker (1.63 g cm−3) and calf thymus DNA (1.43 g cm−3). During sucrose density gradient centrifugation, the hybrid sedimented slightly faster than mouse tRNA marker. Evidence for the double-stranded nature of the hybrid was found by thermal denaturation of the complex. The RNA of the hybrid became increasingly sensitive to ribonuclease as the hybrid was heated at successively higher temperatures.

scholarly journals Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina

1982 ◽  
Vol 203 (3) ◽  
pp. 571-575 ◽  
Author(s):  
T Tahara ◽  
Y Maeda ◽  
A Kuroiwa ◽  
K Ueno ◽  
M Obinata ◽  
...  
Keyword(s):  
Density Gradient ◽  
Storage Protein ◽  
Messenger Rna ◽  
Fat Body ◽  
Density Gradient Centrifugation ◽  
Signal Sequence ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation

Storage-protein mRNA was found to be abundant in poly(A)-containing RNA extracted from the fat-body of third-instar larvae of Sarcophaga peregrina (fleshfly). This RNA sedimented at the position of 19S on sucrose-density-gradient centrifugation and the product of its translation in vitro was 75K protein (protein of mol.wt. 75 000), which was precipitated specifically with antibody against storage protein. This product was suggested to contain a signal sequence that is missing in mature storage protein. The poly(A)-containing RNA was also found to contain much of another mRNA coding for 25K protein (protein of mol.wt. 25 000), but the function of this protein is unknown.

Further evidence for the presence of androgen receptors in the lungs and in the head appendages of the cock

1977 ◽  
Vol 55 (3) ◽  
pp. 263-265 ◽  
Author(s):  
Jean Y. Dubé ◽  
Pierre Chapdelaine ◽  
Roland R. Tremblay
Keyword(s):  
Density Gradient ◽  
Mechanism Of Action ◽  
Androgen Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
In Vitro Binding ◽  
Vitro Binding

The presence of cytoplasmic dihydrotestosterone receptors in the lungs, the comb, the wattle, and the ear lobes of the cock was demonstrated by sucrose density-gradient centrifugation. All these tissues exhibited saturable 'in vitro' binding of dihydrotestosterone in the 8–11S region of the gradient. When 0.5 M KCl was added to the lung, wattle, and comb cytosols and to the gradients, the radioactive dihydrotestosterone migrated in the 4–5S region. These studies suggest that the mechanism of action of androgens in the head appendages of the cock and in other target tissues is similar.

Study of tobacco mosaic virus in vitro disassembly by sucrose density gradient centrifugation and agarose gel electrophoresis

1984 ◽  
Vol 62 (3) ◽  
pp. 457-462 ◽  
Author(s):  
Richard Hogue ◽  
Alain Asselin
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Density Gradient ◽  
Gel Electrophoresis ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Sucrose Density Gradient Centrifugation

In vitro disassembly of tobacco mosaic virus (TMV) strains U1, U2, U4, U6, and U7 with alkali and urea was studied by sucrose or sucrose–dimethylsulfoxide (DMSO) density gradient centrifugation and by agarose gel electrophoresis. All strains gave similar decapsidation patterns with both agents when partially stripped virus particles (PSVs) were analyzed by sedimentation and electrophoresis. However, U6 was more sensitive to decapsidation than the other strains and U2 exhibited resistance to decapsidation. Agarose gel electrophoresis of TMV decapsidation products allowed the detection of several classes of PSVs in addition to aggregation products involving PSVs and monomer particles. Agarose gel electrophoresis is thus very rapid and useful for analysis of TMV disassembly products especially when aggregation phenomena and kinetic studies with numerous samples are considered.

scholarly journals FORMATION OF MITOCHONDRIA IN NEUROSPORA CRASSA

1963 ◽  
Vol 16 (3) ◽  
pp. 483-499 ◽  
Author(s):  
David J. L. Luck
Keyword(s):  
Neurospora Crassa ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Continuous Process ◽  
Sucrose Density Gradient ◽  
Mitochondrial Fraction ◽  
Sucrose Density Gradient Centrifugation ◽  
Mitochondrial Mass ◽  
Logarithmic Growth

Cells of a choline-requiring mutant of Neurospora crassa, labeled with radioactive choline, were transferred to unlabeled medium. At various times during their subsequent logarithmic growth, a highly purified mitochondrial fraction was prepared by sucrose density gradient centrifugation, and the distribution of label among individual mitochondria was determined by quantitative autoradiography. Preliminary experiments indicated that, under the conditions of this "washout" experiment, choline served as a stable mitochondrial label. Radioautographic analysis showed that, in fully labeled mycelia and for three mass doubling cycles in the unlabeled medium, radioactivity was randomly distributed among all mitochondria; i.e., the distribution of autographic grains among individual mitochondria followed a Poisson distribution. In experiments in which pulse labeling for 10 minutes was used, the label was randomly distributed among all mitochondria. The data suggest that the mitochondrial mass is increased by a continuous process of addition of new lecithin units to the already existing mitochondrial framework.

The isolation and purification of endophyte DNA from Alnus glutinosa nodules

1983 ◽  
Vol 61 (11) ◽  
pp. 2855-2858 ◽  
Author(s):  
Beth C. Mullin ◽  
Priyavadan A. Joshi ◽  
Chung Sun An
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Alnus Glutinosa ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Isolation And Purification ◽  
Total Dna ◽  
Nodule Tissue

Total DNA was extracted from leaves, roots, and nodules of Alnus glutinosa seedlings. Leaf and root DNA was shown to have a buoyant density in CsCl of ~ 1.68 with no satellite bands at a higher density. The endophyte DNA, which has a much greater buoyant density (ρ = ~ 1.72), was separated from the plant nodule DNA by centrifugation in CsCl. The yield of endophyte DNA was much greater when DNA was extracted directly from nodule tissue than when it was extracted from endophyte which had been isolated from nodules by sucrose density gradient centrifugation.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

Sign in / Sign up

Export Citation Format

Share Document