Conformation parameters of linear macromolecules from velocity sedimentation and other hydrodynamic methods

Methods ◽  
2011 ◽  
Vol 54 (1) ◽  
pp. 124-135 ◽  
Author(s):  
Georges M. Pavlov ◽  
Igor Y. Perevyazko ◽  
Olga V. Okatova ◽  
Ulrich S. Schubert
Keyword(s):  
Velocity Sedimentation ◽  
Linear Macromolecules

SELECTIVE ISOLATION OF HUMAN X-BEARING SPERM BY DIFFERENTIAL VELOCITY SEDIMENTATION IN PERCOLL DENSITY GRADIENTS

1984 ◽  
Vol 5 (2) ◽  
pp. 187-194 ◽  
Author(s):  
SATORU KANEKO ◽  
SHIGERU OSHIO ◽  
TOSHIFUMI KOBAYASHI ◽  
HIDEO MOHRI ◽  
RIHACHI IIZUKA
Keyword(s):  
Velocity Sedimentation ◽  
Density Gradients ◽  
Selective Isolation

A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 595-605 ◽  
Author(s):  
Bradley J Merrill ◽  
Connie Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Synthetic Lethality ◽  
Velocity Sedimentation ◽  
Recombinational Repair ◽  
Repair Pathway ◽  
Dna Breaks ◽  
Single Stranded Dna ◽  
Double Stranded Dna

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

Separation By Velocity Sedimentation of Human Haemopoietic Precursors Forming Colonies in vivo and in vitro Cultures

1985 ◽  
Vol 18 (4) ◽  
pp. 399-406
Author(s):  
E. Niskanen ◽  
J. R. Wells ◽  
D. W. Golde ◽  
M. J. Cline
Keyword(s):  
In Vitro Cultures ◽  
Velocity Sedimentation

scholarly journals Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane

1978 ◽  
Vol 77 (3) ◽  
pp. 722-734 ◽  
Author(s):  
MM Weiser ◽  
MM Neumeier ◽  
A Quaroni ◽  
K Kirsch
Keyword(s):  
Cell Surface ◽  
Atpase Activity ◽  
Surface Membrane ◽  
Basal Membrane ◽  
Crypt Cell ◽  
Velocity Sedimentation ◽  
Intact Cells ◽  
Microvillus Membrane ◽  
Villus Cell ◽  
Crypt Cells

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.

ULTRACENTRIFUGE AND DIFFUSION STUDIES ON GLUTEN

1942 ◽  
Vol 20c (3) ◽  
pp. 130-159 ◽  
Author(s):  
A. G. McCalla ◽  
Nils Gralén
Keyword(s):  
Sodium Salicylate ◽  
Minimum Weight ◽  
Average Molecular Weight ◽  
Sedimentation Equilibrium ◽  
Velocity Sedimentation ◽  
Molecular Characteristics ◽  
Weight Average Molecular Weight ◽  
Diffusion Constants ◽  
Diffusion Studies ◽  
And Diffusion

The molecular characteristics of gluten in sodium salicylate solutions were studied by means of sedimentation velocity, sedimentation equilibrium, and diffusion measurements. The proportion of total gluten protein molecularly dispersed increased with increase in concentration of sodium salicylate up to 12%, but the dispersed portions had essentially the same sedimentation constant (2.5 ± 0.15) regardless of the concentration of the dispersing medium.The most soluble 25 per cent of the gluten was all molecularly dispersed, but was definitely inhomogeneous. The weight-average molecular weight of this fraction was 44,000, but there is reason to believe the minimum weight may be about 35,000. None of the other fractions was entirely molecularly dispersed, the proportion decreasing with decreasing solubility of the fractions. Aggregates of many sizes existed in all of these fractions, but only the most insoluble contained aggregates large enough to cause opacity. Sedimentation constants of the molecularly dispersed portions increased slightly with decreasing solubility, while diffusion constants decreased markedly. None of the fractions yielded normal curves (diffusion diagrams) but the more soluble the fraction, the more nearly normal the curve. The inhomogeneity responsible for the varying rates of diffusion was due partly to differences in proportion and properties of the molecularly dispersed gluten and partly to aggregates.All properties showed progressive changes both within and between the arbitrarily produced fractions. These results, therefore, support the hypothesis that gluten is a protein system showing progressive and regular changes in properties with change in solubility.

scholarly journals Far3 and Five Interacting Proteins Prevent Premature Recovery from Pheromone Arrest in the Budding Yeast Saccharomyces cerevisiae

2003 ◽  
Vol 23 (5) ◽  
pp. 1750-1763 ◽  
Author(s):  
Hilary A. Kemp ◽  
George F. Sprague,
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Open Reading Frames ◽  
Velocity Sedimentation ◽  
Interacting Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cycle Arrest ◽  
Mutant Cells ◽  
Two Hybrid ◽  
Reading Frames

ABSTRACT In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G1 arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.

Mouse cells contain two distinct ras gene mRNA species that can be translated into a p21 onc protein

1982 ◽  
Vol 2 (11) ◽  
pp. 1339-1345
Author(s):  
R W Ellis ◽  
D DeFeo ◽  
M E Furth ◽  
E M Scolnick
Keyword(s):  
Normal Mouse ◽  
Velocity Sedimentation ◽  
Ras Gene ◽  
Mrna Species ◽  
P21 Ras ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Mouse Cells ◽  
Murine Sarcoma

The Kirsten (Ki) and Harvey (Ha) strains of murine sarcoma virus encode a 21,000-dalton protein (p21 ras) which is the product of the transforming gene of these viruses. Normal cells express low levels of p21 ras encoded by cellular genes (Ki-ras and Ha-ras) homologous to the Ki and Ha murine sarcoma virus transformation genes. A bone marrow-derived mouse cell line, 416B, has been shown to express unusually high levels of p21 ras. In this manuscript, we investigated the molecular biology of p21 ras gene expression in 416B and other normal mouse cells. We identified four distinct polyadenylated and polysome-associated RNAs, two related to Ki-ras and two to Ha-ras. The levels in 416B cells of the two Ki-ras RNAs, sized 5.2 and 2.0 kilobases, were both elevated approximately 25-fold over levels found in normal mouse cells; there was no corresponding change in 416B cells in the levels of the two Ha-ras RNAs. We partially purified the two Ki-ras mRNAs and separated them by velocity sedimentation in sucrose density gradients. Both the 5.2- and 2.0-kilobase mRNAs could be translated in vitro into p21 ras. These results show that a cellular onc protein can be translated from two distinct cellular mRNA species.

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