The RNA Fractions of Chromatin from L-Cells

1974 ◽  
Vol 52 (10) ◽  
pp. 922-935 ◽  
Author(s):  
John J. Monahan ◽  
Ross H. Hall
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Weight Fraction ◽  
Polyacrylamide Gel ◽  
Low Molecular Weight ◽  
Two Dimensional ◽  
Cell Nuclei ◽  
L Cell

Chromatin obtained from L-cell nuclei has been shown to contain three distinct RNA fractions. One, a low molecular weight RNA fraction (3–7 S), has been isolated and purified. Two-dimensional polyacrylamide gel electrophoresis indicates that this fraction contains at least 28 diverse RNA components. Both the rate of incorporation of [32P]H3PO4 into this fraction and the loss of [3H]uridine from the fraction after a 1 h pulse suggest that these RNA's are slowly synthesized and are very stable. Not all the species within the low molecular weight fraction have the same turnover rates.A high molecular weight RNA fraction (15–30 S) has also been isolated from L-cell chromatin. From the rate of [32P]H3PO4 incorporation and the loss of [3H]uridine from this fraction after a 1 h pulse, it appears that this RNA fraction is rapidly synthesized and is either unstable or is rapidly removed from chromatin once synthesized.A third RNA fraction also present in L-cell chromatin appears to be tightly bound to the DNA. It has been isolated and shown to be of low molecular weight (3–5 S). This RNA also appears to be rapidly synthesized and to be metabolically unstable.

scholarly journals Mapping of the genes controlling high-molecular-weight glutelin subunits of rye on the long arm of chromosome 1R

1984 ◽  
Vol 44 (2) ◽  
pp. 117-123 ◽  
Author(s):  
N. K. Singh ◽  
K. W. Shepherd
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Translocation Line ◽  
Sds Page ◽  
Chromosome 1R

SUMMARYThe gene(s) controlling the high-molecular-weight glutelin subunits in rye (designated as Glu-Rl) was mapped with respect to the centromere using a 1RL-1DS wheat-rye translocation line and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Analysis of 479 seeds from test-crosses between a 1R/1RL-1DS heterozygote and the cultivar India 115, revealed 14·6% aneuploid and 3·95% recombinant progeny. Excluding the aneuploids, this locus was calculated to be 4·65 ± 1·04 cM from the centromere on the long arm of chromosome 1R, which is comparable to the position of the homoeologous loci in wheat and barley.

Fractionation of Chromatin Components

1973 ◽  
Vol 51 (5) ◽  
pp. 709-720 ◽  
Author(s):  
John J. Monahan ◽  
Ross H. Hall
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Urea Solution ◽  
Equilibrium Density ◽  
Tissue Culture Cell ◽  
Low Molecular Weight ◽  
Nonhistone Proteins

A general method for isolation and fractionation of chromatin into its four major components, DNA, RNA, histories, and nonhistone proteins, is described. The procedure avoids the use of strongly acidic or alkaline conditions, or the use of ionic detergents or phenol. As few as 14 × 106 cells can be used. The procedure is reasonably rapid and has been used successfully with a number of tissue culture cell lines. The chromatin components are dissociated in a 3 M NaCl – 5 M urea solution containing 2-mercaptoethanol and EDTA. The DNA and high molecular weight RNA are collected by high-speed centrifugation and DNA is separated from the RNA by means of Cs2SO4 equilibrium density centrifugation. The histones, nonhistone proteins, and low molecular weight RNA's are fractionated using DEAE-cellulose column chromatography and polyacrylamide gel electrophoresis. A small amount (< 1%) of protein is present in the DNA and RNA fractions. At least 11 low molecular weight RNA subfractions can be detected by means of polyacrylamide gel electrophoresis.

Analysis of proteins in rabbit pulmonary surfactant using monoclonal antibodies

1986 ◽  
Vol 250 (3) ◽  
pp. C460-C467 ◽  
Author(s):  
R. J. King ◽  
H. M. Martin ◽  
J. B. Baseman ◽  
J. Morrison-Plummer
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Pulmonary Surfactant ◽  
Polyacrylamide Gel Electrophoresis ◽  
Low Molecular Weight ◽  
Weight Group ◽  
Molecular Weights ◽  
Intermediate Group

We have used monoclonal antibodies developed against the apolipoproteins associated with pulmonary surfactant purified from rabbit lavage fluid to study the expression of epitopes common to these proteins. The pulmonary surfactant contained nearly 20 proteins, of which at least 10 were not derived from serum. Electrophoresis, with sulfhydryl reduction of these proteins indicated apparent molecular weights of approximately 155, 135, 125, and 115 X 10(3) (high-molecular-weight group); 80, 70, and 60 X 10(3) (intermediate group); and 18 through 10 X 10(3) (low-molecular-weight group). Two-dimensional polyacrylamide gel electrophoresis, in which the proteins were electrophoresed without reduction in the first dimension, but with sulfhydryl reduction in the second dimension, revealed that the 80, 70, and 60 X 10(3) proteins dissociated into proteins of nominal molecular weights of 40, 35, and 30 X 10(3), respectively. In contrast, the 125 and 115 X 10(3) proteins of the high-molecular-weight group contained a protein which could only be reduced to a minimum molecular weight of 55 to 60 X 10(3). Monoclonal antibodies generally were of three types: those that reacted strongly with the high-molecular-weight group and weakly with the intermediate group; those that reacted conversely; and those that reacted only with the low-molecular-weight group. Our results indicate that at least two different surfactant apolipoproteins, with differing minimum molecular weights in SDS-polyacrylamide gel electrophoresis, have common epitopes. Although these results cannot certify a physiological relationship between these proteins, they suggest that the intracellular synthesis or extracellular processing of surfactant apolipoproteins may be more complicated than predicted by the findings of previous experiments, perhaps involving the posttranslational assembly of one surfactant protein into oligomers which resist dissociation under the conditions used for the analyses.

Method for measuring the concentration of urinary proteins according to their molecular size category.

1976 ◽  
Vol 22 (5) ◽  
pp. 667-672 ◽  
Author(s):  
A J Pesce ◽  
A Hsu ◽  
C Kornhauser ◽  
K Sethi ◽  
B S Ooi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Low Molecular Weight ◽  
Tubular Proteinuria ◽  
Equal Concentration ◽  
Glomerular Proteinuria ◽  
Low Molecular Weight Proteins

Abstract We combined the use of a concentrating device (Minicon) and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to semi-quantitate the concentration of (a) the collective low-molecular-weight proteins and (b) of albumin excreted in the urine of patients after renal transplantation. Analytical recovery of many serum proteins from samples concentrated 100-fold in the Minicon apparatus was about 70%. It was possible to examine many urine samples by polyacrylamide gel electrophoresis after concentration with this device. The reproducibility (CV) of the technique was on the order of 20% when albumin and low-molecular-weight protein were in about equal concentration. The method was adequate to differntiate glomerular and tubular proteinuria, because in glomerular proteinuria the ratio of albumin to low-molecular-weight proteins is about 20/1, whereas in tubular proteinuria the ratio is about 1/1.

scholarly journals Additional glycoprotein defects in Bernard-Soulier's syndrome: confirmation of genetic basis by parental analysis

Blood ◽  
1983 ◽  
Vol 62 (4) ◽  
pp. 800-807 ◽  
Author(s):  
MC Berndt ◽  
C Gregory ◽  
BH Chong ◽  
H Zola ◽  
PA Castaldi
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Membrane Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Membrane Glycoproteins ◽  
Glycoprotein Ib ◽  
Membrane Complex ◽  
Platelet Protein

Abstract The glycoprotein profile of Bernard-Soulier platelets was examined by labeling washed platelets with periodate 3H-sodium borohydride, a procedure that labels greater than 30 glycoproteins on the membrane surface of normal platelets. Three Bernard-Soulier patients were studied; two were siblings and the third was unrelated. The platelet protein and glycoprotein profiles were evaluated under nonreduced and reduced conditions using 5%-15% exponential SDS-polyacrylamide gel electrophoresis. The two siblings completely lacked glycoprotein Ib (GPIb). The unrelated patient had congruent to 7% of the normal level. This was confirmed by two-dimensional nonreduced-reduced SDS- polyacrylamide gel electrophoresis, a procedure that allows clear separation of the disulfide-linked subunits of GPIb, GPIb alpha (mol wt 145,000), and GPIb beta (mol wt 25,000) from other membrane glycoproteins. On one-dimensional analysis, Bernard-Soulier's syndrome (BSS) platelets also lacked the peripheral membrane glycoprotein, GPV (mol wt 82,000) and a low molecular weight glycoprotein, GPIX, (nonreduced or reduced, mol wt congruent to 22,000). The two- dimensional gel system also revealed the absence of a minor glycoprotein with a molecular weight of congruent to 100,000 (GP 100). Quantitation of these proteins solubilized from electrophoretograms showed that the siblings' parents had congruent to 50% levels of GPIb, GPIX, and GP 100. A monoclonal antibody against glycoprotein Ib, FMC 25, was negative by immunofluorescence against Bernard-Soulier platelets and immuneprecipitated both GP Ib and GPIX from Triton X100 solubilized, labeled platelets. The combined results suggest that the apparent genetic absence of multiple proteins in Bernard-Soulier platelets is due, in part, to the presence in normal platelets of a tight membrane complex between glycoprotein Ib and at least one of the other absent glycoproteins.

scholarly journals Arterial basement-membrane-like material isolated and characterized from rabbit aortic myomedial cells in culture

1983 ◽  
Vol 211 (2) ◽  
pp. 397-404 ◽  
Author(s):  
L Heickendorff ◽  
T Ledet
Keyword(s):  
Molecular Weight ◽  
Basement Membrane ◽  
High Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Type I

Arterial basement-membrane-like material was isolated from rabbit aortic myomedial cell cultures by sonication and differential centrifugation. Isolated basement-membrane-like material was shown to be free of both cellular and matrix contaminants, on the basis of determinations of DNA, RNA, cholesterol, phosphorus and (Na+ + K+)-activated ATPase, combined with electron microscopy. Amino acid analyses showed that arterial basement-membrane-like material was composed of predominantly non-collagenous amino acids. Evaluated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, reduced basement-membrane-like material comprised six major and about 30 minor components in the Mr range 10 000-600 000. One of the major peptides (Mr 225 000) was disulphide-linked. Periodic acid-Schiff staining of gels indicated that most high-molecular-weight components were glycoproteins. Two-dimensional gel electrophoresis resolved reduced basement-membrane-like material into more than 100 components, with pI from 5 to 7. The disulphide-linked Mr-225 000 peptide appeared heterogeneous, with pI of 5.6-6.0, and was considered to represent fibronectin. All major peptides were of non-collagenous nature, on the basis of their susceptibility to pepsin and resistance to collagenase. Purified myomedial basement-membrane-like material contained collagenous peptides, as indicated by the presence of hydroxyproline and hydroxylysine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pepsin-treated and reduced basement-membrane-like material revealed five high-molecular-weight collagenous components appearing in the Mr range 105 000-375 000 relative to type I collagen standards.

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