scholarly journals Evidence of low molecular weight RNAs involved in permanent character changes in amoebae

1980 ◽  
Vol 43 (1) ◽  
pp. 329-340
Author(s):  
S.E. Hawkins
Keyword(s):  
Microsomal Fraction ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Material ◽  
Sucrose Density Gradient ◽  
Low Molecular Weight ◽  
Sucrose Density Gradient Centrifugation ◽  
Rna Molecules ◽  
Molecular Weights ◽  
Lowered Sensitivity

Previous studies have established that ‘informational molecules’ present in cytoplasmic fractions of A. discoides may be transferred by microinjection into A. proteus. Clones derived from injected cells showed various changers, including lowered sensitivity to growth in streptomycin and neomycin, in which respects they resembled A. discoides. These changes in response to antibiotics were transferred independently and were permanent, the information being replicated over many generations. The most ‘active’ material in terms of the number of clones showing character changes was found following injection of 16S ribonucleoprotein obtained after sucrose density gradient centrifugation of the mcirosomal fraction. Polyacrylamide gel electrophoresis of the 16S material showed 3 small peaks of RNA. In order to obtain adequate amounts of material, these peaks of RNA were identified in electrophoresis profiles of RNA extracted from the whole microsomal fraction, and RNA eluted from these latter gels was injected into A. proteus. Although the number of surviving clones was low, all were examined for their response to growth in either streptomycin, neomycin, erythromycin or chloroquine. After injection of RNA eluted from the 3 small peaks of RNA (slices 26–33), 8 out of 10 and 9 out of 10 clones showed lowered sensitivity to growth in streptomycin and neomycin respectively, and resembled the donor A. discoides. No changes in responses to antibiotics were obtained from clones derived from cells injected with RNA eluted from another region of the gel, or after ribonuclease treatment of the RNA from slices 26–33. The relative molecular weights of these ‘informational’ RNA molecules were found to be between 9 and 13 X 10(4) Daltons.

scholarly journals Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)

1973 ◽  
Vol 135 (1) ◽  
pp. 73-79 ◽  
Author(s):  
J. F. Giorgini ◽  
F. L. De Lucca
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Dimethyl Sulphoxide ◽  
Low Molecular Weight ◽  
28S Rrna ◽  
Sucrose Density Gradient Centrifugation ◽  
Crotalus Durissus Terrificus ◽  
Crotalus Durissus

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ‘6S’ and ‘8S’ low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0°C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.

scholarly journals Chloroplast and cytoplasmic low-molecular-weight ribonucleic acid components of the leaf of Vicia faba L.

1968 ◽  
Vol 106 (3) ◽  
pp. 689-698 ◽  
Author(s):  
T. A. Dyer ◽  
Rachel M. Leech
Keyword(s):  
Molecular Weight ◽  
Nucleic Acids ◽  
Serum Albumin ◽  
Vicia Faba ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Low Molecular Weight ◽  
Sucrose Density Gradient Centrifugation ◽  
Vicia Faba L

1. A method for the extraction of plant nucleic acids and their separation on methylated-serum-albumin–kieselguhr columns is described. It is demonstrated that the characteristics of the elution profiles of material from the same source are consistently reproducible. 2. Major dissimilarities were found in the elution profiles of nucleic acids from root and from leaves of Vicia faba L. These dissimilarities were confirmed by polyacrylamide-gel electrophoresis. 3. Four distinct types of low-molecular-weight RNA were demonstrated to be present in leaves, clearly distinguished by their behaviour when chromatographed on methylated-serum-albumin–kieselguhr columns. (a) Both cytoplasmic and chloroplast ribosomes contained a low-molecular-weight RNA, and these components were distinct from each other. (b) The chloroplast possessed a unique ‘soluble’ RNA (i.e. RNA that is not precipitated by centrifugal forces that sediment ribosomes) which was not present in the rest of the cell. (c) A soluble component, probably transfer RNA, was found in both the chloroplasts and in the cytoplasm. 4. The components distinguishable by methylated-serum-albumin–kieselguhr column chromatography could not be distinguished by sucrose-density-gradient centrifugation.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

scholarly journals Isolation of 10-nm filaments from astrocytes in the mouse optic nerve.

1981 ◽  
Vol 88 (1) ◽  
pp. 245-250 ◽  
Author(s):  
S Tsukita ◽  
H Ishikawa ◽  
M Kurokawa
Keyword(s):  
Optic Nerve ◽  
Peptide Mapping ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Sucrose Density Gradient Centrifugation ◽  
Optic Nerves ◽  
10 Nm Filaments

Astroglial filaments approximately 10 nm in diameter were isolated from degenerated mouse optic nerves by Triton X-100 and DNase I treatments followed by sucrose density gradient centrifugation. 2-4 wk after bilateral enucleation, optic nerves contained virtually a single population of 10-nm filaments (astroglial filaments), free from neurofilaments. In negative-staining and thin-section electron microscopy, the isolated filaments were seen as nonbranching linear structures with smooth contour, and were morphologically identical to those in situ. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the isolated filaments to be composed of two major polypeptides with molecular weights of 45,000 and 55,000, present in an approximate molar ratio of 1:1. These findings, together with the results of one-dimensional peptide mapping and solubility study, indicate that the astroglial filaments in the mouse optic nerve are primarily composed of these two polypeptides.

scholarly journals Assembly of immunoglobulin M. Blocked thiol groups of intracellular 7S subunits

1971 ◽  
Vol 123 (4) ◽  
pp. 629-634 ◽  
Author(s):  
Brigitte A. Askonas ◽  
R. M. E. Parkhouse
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Reducing Agent ◽  
Immunoglobulin M ◽  
Cysteine Residues ◽  
Sucrose Density Gradient Centrifugation ◽  
Spontaneous Polymerization ◽  
Low Protein ◽  
Mouse Myeloma

We have shown previously that immunoglobulin M (IgM) is present within IgM-forming cells mainly in its 7S subunit form (IgMs), whereas only fully assembled IgM pentamers are secreted. There is no spontaneous polymerization of intracellular IgMs in cell lysates, suggesting that the 7S subunits had blocked cysteine residues. This suggestion was explored and confirmed in the present paper. Radioactive IgM (secreted) and IgMs (intracellular) were prepared by sucrose-density-gradient centrifugation after incubation of cells of the IgM-producing mouse myeloma MOPC 104E with [3H]leucine. We investigated the susceptibility to reduction of fully assembled mouse IgM and its reconstitution from subunits by analysis by polyacrylamide-gel electrophoresis under dissociating conditions. With increasing concentrations of dithioerythritol, interchain disulphide bonds were cleaved in the following order: inter-IgMs subunit, intra-IgMs subunit H-H, intra-IgMs subunit H-L. Removal of the reducing agent from IgM-reduction mixtures by filtration through Sephadex G-25 caused partial reconstitution of IgM at low protein concentrations (5–100μg/ml) and total reconstitution at higher protein concentrations (300μg/ml or more). Isolated radioactive intracellular IgMs showed no tendency to polymerize unless first treated with a reducing agent; under optimum conditions removal of the reducing agent caused 70% of the subunits to be assembled into IgM. Similar assembly occurred when IgMs was isolated from cells that had been lysed in the presence of an irreversible alkylating reagent (iodoacetamide). The intracellular IgMs cysteine residues responsible for inter-IgMs linkage therefore appear to be reversibly blocked within the cells. Assembly into IgM is thus controlled by removal of this block during secretion.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

scholarly journals Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

1970 ◽  
Vol 117 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Keitaro Kato ◽  
Hiroyuki Ide ◽  
Tsuranobu Shirahama ◽  
William H. Fishman
Keyword(s):  
Endoplasmic Reticulum ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Mouse Kidney ◽  
Differential Centrifugation ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Glucuronidase Activity

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-14C]glucosamine or l-[U-14C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.

Ribonucleic acids of differentiating and non-differentiating uredosporelings of wheat stem rust

1974 ◽  
Vol 52 (6) ◽  
pp. 1309-1317 ◽  
Author(s):  
W. K. Kim ◽  
R. Rohringer
Keyword(s):  
Ribosomal Rna ◽  
Stem Rust ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Wheat Stem Rust ◽  
Sucrose Density Gradient Centrifugation ◽  
Ribonucleic Acids ◽  
Molecular Weights ◽  
Germ Tubes

Uredospores of wheat stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn.) were deposited onto Millipore membranes and allowed to germinate. Those remaining continuously at 20° formed germ tubes only (non-differentiated), but those exposed to 30° for 90 min after the first 2 h of germination developed infection structures corresponding to appressoria and substomatal vesicles (differentiated).Nucleic acids were extracted with a phenol method from resting uredospores and from differentiated and non-differentiated sporelings. The amount of extractable RNA decreased as germination progressed, but no RNA was detected in the germination medium. The decrease in extractable RNA (up to 40%) occurred in both differentiated and non-differentiated sporelings.Acrylamide gel electrophoresis was used to separate RNA species and to determine their approximate molecular weights (in daltons): sporelings contained 25-S (1.65 × 106) and 18-S (0.80 × 106) ribosomal RNA (rRNA), 5-S (3.6 × 104) rRNA, and 4.5-S (2.4 × 104) transfer RNA (tRNA). Radioactive uridine, fed to sporelings, was incorporated mostly into 5-S rRNA and (or) tRNA.Acrylamide gel electrophoresis and sucrose density gradient centrifugation revealed that differentiated sporelings contained a type of RNA that was not detected in non-differentiated sporelings. It was heterogeneous and migrated in the 16-S to 5-S interval on polyacrylamide gels. Some of the RNA present in this fraction may have been preformed in resting spores and released from more complex material during the process of differentiation.

scholarly journals Viroids as Companions of a Professional Career

Viruses ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 245 ◽  
Author(s):  
Núria Duran-Vila
Keyword(s):  
Tertiary Structure ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Critical Role ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Biological Assays ◽  
Professional Career ◽  
Staining Methods ◽  
Relationship Of

Since the early 1970s when “virus-like” agents were considered as the cause of two diseases (potato spindle tuber and citrus exocortis), their study and further characterization have been linked to the development and use of molecular biology tools. Sucrose density gradient centrifugation and polyacrylamide gel electrophoresis (PAGE) played a critical role in the pioneering studies of PSTVd and citrus exocortis viroid (CEVd). This was later modified by using other PAGEs (sequential PAGE, return PAGE, two-dimensional PAGE), and/or different staining methods (ethidium bromide, silver nitrate, etc.). Since then, disease-causing agents suspected to be viroids were usually subjected to a number of tests to define their: (i) Molecular nature (RNA or DNA; single stranded or double stranded; circular or linear RNA); (ii) molecular weight; (iii) secondary and tertiary structure. Further biological assays are also essential to establish the relationship of a viroid with plant disease and to fulfill Koch’s postulates.

scholarly journals Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes.

1975 ◽  
Vol 65 (3) ◽  
pp. 577-586 ◽  
Author(s):  
J Noseworthy ◽  
G H Smith ◽  
S R Himmelhoch ◽  
W H Evans
Keyword(s):  
Guinea Pig ◽  
Polymorphonuclear Leukocytes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Species Difference ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Supernatant Fraction ◽  
Sucrose Density Gradient Centrifugation ◽  
Electrophoretic Patterns

The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented.

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