scholarly journals The effect of reaction with formaldehyde on the sedimentation rates of ribonucleic acids

1968 ◽  
Vol 107 (6) ◽  
pp. 851-859 ◽  
Author(s):  
M. L. Fenwick
Keyword(s):  
Hydrogen Bonding ◽  
Ribosomal Rna ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Linear Structure ◽  
Sedimentation Rates ◽  
Amino Groups ◽  
Ribonucleic Acids ◽  
Molecular Weights ◽  
Virus Rna

It has been reported that the RNA of several bacteriophages and that of the larger ribosomal sub-units of mammalian cells sediment faster in the presence of 0·1m-sodium chloride than is expected from their estimated molecular weights. The effect of blocking the hydrogen-bonding amino groups of these and other types of RNA was studied. The RNA of phage R17 no longer sedimented anomalously fast after treatment with formaldehyde. In contrast, the larger ribosomal RNA of HeLa cells appeared more aberrant than before, sedimenting faster than tobacco-mosaic-virus RNA (mol.wt. 2×106) in the presence of formaldehyde. The rapidly labelled nuclear 45s RNA of HeLa cells still sedimented faster than the larger ribosomal RNA after reaction with formaldehyde, showing no evidence of disaggregation. It is suggested that both the large ribosomal RNA and the 45s RNA of HeLa cells may have a non-linear structure.

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.

Acid Soluble Nucleotides and Ribonucleic Acids from Germinating Jack Pine Seeds

1972 ◽  
Vol 2 (3) ◽  
pp. 206-216 ◽  
Author(s):  
D. J. Durzan ◽  
J. Pitel ◽  
P. K. Ramaiah
Keyword(s):  
Ribosomal Rna ◽  
Adenine Nucleotides ◽  
Unit Weight ◽  
Polyacrylamide Gels ◽  
Electron Microscopic ◽  
Ribonucleic Acids ◽  
Total Rna ◽  
Molecular Weights ◽  
Electrophoretic Mobilities ◽  
Pine Seedlings

In germinating pine seedlings, acid soluble nucleotides, initially rich in AMP, accumulated, and were increasingly dominated by ATP. By 7 days, when hypocotyls were green but still dependent on the female gametophyte, the percentage of adenine nucleotides declined as other nucleotides increased. At 11 days, when gametophytic reserves were depleted, and seedlings were exposed to 32P- phosphoric acid, adenine nucleotides accounted for over 70% of the recovery of 32P from the nucleotide fraction.In seedlings, total RNA per unit weight increased to the 6th day, then levelled off. Comparison of electrophoretic mobilities of pine RNA on 2.5% polyacrylamide gels with ribosomal RNA from other sources revealed the following main types based on estimated molecular weights: 1.3, 1.1, 0.7, and 0.56 × 106 daltons, corresponding to 25S, 23S, 18S, and 16S, respectively. On 10% polyacrylamide gels, the mobility of low molecular weight pine RNA was similar to that of yeast tRNA. The 1.3 and 0.7 × 106 dalton fractions, representing ribosomal RNA, contributed 60–90% of the total RNA. During germination, the ratio of 1.3 to 0.7 × 106 RNA dropped from 2.4 to 1.7 and reciprocated the pattern for water content of the seedling.32P was incorporated mainly into the 25S and 18S RNA by 11-day-old seedlings, indicating that synthesis of ribosomes was a major event in growing seedlings. During germination, the synthesis of RNA corresponded to an increase of total RNA detected cytochemically and to an increase of new cytoplasm with more ribosomes, as observed previously with hypocotyl cells by electron microscopic procedures.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

scholarly journals Identification and Functional Mapping of the Mycoplasma fermentans P29 Adhesin

2002 ◽  
Vol 70 (9) ◽  
pp. 4925-4935 ◽  
Author(s):  
Spencer A. Leigh ◽  
Kim S. Wise
Keyword(s):  
Disulfide Bond ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Flow Cytometric Analysis ◽  
Phase Variation ◽  
Surface Proteins ◽  
Mycoplasma Species ◽  
Phase Variable ◽  
Binding Region ◽  
Mycoplasma Fermentans

ABSTRACT Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein

1992 ◽  
Vol 12 (1) ◽  
pp. 164-171
Author(s):  
M J Matunis ◽  
W M Michael ◽  
G Dreyfuss
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Predicted Amino Acid Sequence ◽  
Cdna Clones ◽  
Hnrnp Proteins

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

scholarly journals Pathogenesis of shigella diarrhea. VII. Evidence for a cell membrane toxin receptor involving β1 {arrow} 4-linked N-acetyl-D-glucosamine oligomers

1977 ◽  
Vol 146 (2) ◽  
pp. 535-546 ◽  
Author(s):  
GT Keusch ◽  
M Jacewicz
Keyword(s):  
Cell Membrane ◽  
Cholera Toxin ◽  
Liver Cell ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Cell Membranes ◽  
Proteolytic Enzymes ◽  
Cell Monolayer ◽  
Toxin Receptor ◽  
Toxin Uptake

The binding of ShigeUa dysenteriae 1 cytotoxin to HeLa cells in culture and to isolated rat liver cell membranes was studied by means of an indirect consumption assay of toxicity from the medium, or by determination of cytotoxicity to the HeLa cell monolayer. Both liver cell membranes and HeLa cells removed toxicity from the medium during incubation, in contrast to WI-38 and Y-1 mouse adrenal tumor cells, both of which neither bound nor were affected by the toxin. Uptake of toxin was directly related to concentration of membranes added, time,and temperature, and indirectly related to the ionic strength of the buffer used. The chemical nature of the membrane receptor was characterized by using three principal approaches: (a) enzymatic sensitivity; (b) competitive inhibition and (c) receptor blockade studies. The receptor was destroyed by proteolytic enzymes, phospholipases (which markedly altered the gross appearance of the membrane preparation) and by lysozyme, but not by a variety of other enzymes. Of 28 carbohydrate and glycoprotein haptens studied, including cholera toxin and ganglioside, only the chitin oligosaccharide lysozyme substrates, per N-acetylated chitotriose, chitotetraose, and chitopentaose were effective competitive inhibitors. Greatest inhibition was found with the trimer, N, N', N" triacetyl chitotriose. Of three lectins studied as possible receptor blockers, including phytohemagglutinin, concanavalin A, and wheat germ agglutinin, only the latter, which is known to possess specific binding affinity for N, N', N" triacetyl chitotriose, was able to block toxin uptake. Evidence from all three approaches indicate, therefore, existence of a glycoprotein toxin receptor on mammalian cells, with involvement of oligomeric β1{arrow}4-1inked N-acetyl glucosamine in the receptor. This receptor is clearly distinct from the G(M1) ganglioside thought to be involved in the binding of cholera toxin to the cell membrane of a variety of cell types susceptible to its action.

Hydrogen-bonding patterns in bis[2,4,6-triazaniumylcyclohexane-1,3,5-tris(olate)-κ3O,O′,O′′]germanium(IV) tetrachloride hexahydrate

2016 ◽  
Vol 72 (1) ◽  
pp. 28-34
Author(s):  
Christian Neis ◽  
Bernd Morgenstern ◽  
Kaspar Hegetschweiler
Keyword(s):  
Hydrogen Bonding ◽  
Hydration Shell ◽  
Chloride Ions ◽  
Three Dimensions ◽  
Water Molecules ◽  
Amino Groups ◽  
Compound C ◽  
Hydrated Salt ◽  
Phd Thesis ◽  
Hydroxy Groups

A first preliminary report on the crystal structure of a hydrated salt formulated as [Ge(taci)2]Cl4·13H2O (taci is 1,3,5-triamino-1,3,5-trideoxy-cis-inositol) appeared more than 20 years ago [Ghisletta (1994). PhD thesis, ETH Zürich. Switzerland]. At that time it was not possible to discriminate unambiguously between the positions of some of the chloride ions and water O atoms, and disorder was thus postulated. In a new determination, a conclusive scheme of hydrogen bonding proves to be a particularly appealing aspect of the structure. Single crystals of the title compound, C12H30GeN6O64+·4Cl−·6H2O or [Ge(taci)2]2Cl8·12H2O, were grown from an aqueous solution by slow evaporation of the solvent. The two [Ge(taci)2]4+cations exhibit a double-adamantane-type structure with exclusive O-atom coordination and approximateD3dsymmetry. The taci ligands adopt a zwitterionic form with deprotonated hydroxy groups and protonated amino groups. Both cations are hydrogen bonded to six water molecules. The structure of the hydration shell of the two cations is, however, slightly different. The {[Ge(taci)2]·6H2O}4+aggregates are interlinked in all three dimensions by further hydrogen bonds of the types N—H...Cl...H—N, N—H...O(H)2...H—N, (Ge)O...H—O(H)...H—N, N—H...O(H)—H...Cl...H—N, (Ge)O...H—O—H...Cl...H—N, N—H...O(H)—H...Cl...H—(H)O...H—N, (Ge)O...H—O—H...Cl...H—(H)O...H—N and Ge(O)...H—O—H...Cl...H—O—H...O(Ge).

scholarly journals Age-dependent ribosomal DNA variations and their effect on cellular function in mammalian cells

2020 ◽  
Author(s):  
Eriko Watada ◽  
Sihan Li ◽  
Yutaro Hori ◽  
Katsunori Fujiki ◽  
Katsuhiko Shirahige ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Copy Number ◽  
Mammalian Cells ◽  
Bone Marrow Cells ◽  
Budding Yeast ◽  
Rdna Repeat ◽  
Age Dependent ◽  
Old Mice ◽  
Rdna Copy ◽  
Rdna Copy Number

AbstractThe ribosomal RNA gene, which consists of tandem repetitive arrays (rDNA repeat), is one of the most unstable regions in the genome. The rDNA repeat in the budding yeast is known to become unstable as the cell ages. However, it is unclear how the rDNA repeat changes in ageing mammalian cells. Using quantitative analyses, we identified age-dependent alterations in rDNA copy number and levels of methylation in mice. The degree of methylation and copy number of rDNA from bone marrow cells of 2-year-old mice were increased by comparison to 4-week-old mice in two mouse strains, BALB/cA and C57BL/6. Moreover, the level of pre-rRNA transcripts was reduced in older BALB/cA mice. We also identified many sequence variations among the repeats with two mutations being unique to old mice. These sequences were conserved in budding yeast and equivalent mutations shortened the yeast chronological lifespan. Our findings suggest that rDNA is also fragile in mammalian cells and alterations within this region have a profound effect on cellular function.Author SummaryThe ribosomal RNA gene (rDNA) is one of the most unstable regions in the genome due to its tandem repetitive structure. rDNA copy number in the budding yeast increases and becomes unstable as the cell ages. It is speculated that the rDNA produces an “aging signal” inducing senescence and death. However, it is unclear how the rDNA repeat changes during the aging process in mammalian cells. In this study, we attempted to identify the age-dependent alteration of rDNA in mice. Using quantitative single cell analysis, we show that rDNA copy number increases in old mice bone marrow cells. By contrast, the level of ribosomal RNA production was reduced because of increased levels of DNA methylation that represses transcription. We also identified many sequence variations in the rDNA. Among them, three mutations were unique to old mice and two of them were found in the conserved region in budding yeast. We then established a yeast strain with the old mouse-specific mutations and found this shortened the lifespan of the cells. These findings suggest that rDNA is also fragile in mammalian cells and alteration to this region of the genome affects cellular senescence.

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