Wheat embryo ribonucleates. XIV. Mass isolation of mRNA from wheat germ and comparison of its translational capacity with that of mRNA from imbibing wheat embryos

1979 ◽  
Vol 57 (9) ◽  
pp. 1170-1175 ◽  
Author(s):  
A. C. Cuming ◽  
T. D. Kennedy ◽  
B. G. Lane
Keyword(s):  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Wheat Embryo ◽  
Molecular Weights ◽  
Wheat Embryos ◽  
The Subject ◽  
Mass Isolation ◽  
Gradient Fractionation ◽  
Rabbit Reticulocytes

Commercially milled wheat germ is shown to be a convenient source material for facile recovery of mass (milligram) quantities of highly purified poly(A)-rich RNA. This poly(A)-rich RNA is efficiently translated in a nuclease-treated extract of rabbit reticulocytes. By sucrose density gradient fractionation of bulk poly(A)-rich RNA from wheat germ, it has been possible to show that there is a direct relationship between the molecular weights of the polypeptide products of cell-free synthesis and the molecular weights of the wheat mRNA molecules which program their synthesis. As assessed by SDS – polyacrylamide gel electrophoresis, the same array of polypeptides is synthesized when nuclease-treated reticulocyte extract is programmed by poly(A)-rich RNA from either commercially supplied or laboratory-prepared wheat embryos. Significantly, there are gross quantitative if not qualitative differences between the translational capacities of poly(A)-rich RNA from dry and imbibing wheat embryos, and the possible importance of these differences for interpreting a changing pattern of polypeptide synthesis in imbibing wheat embryos is the subject of a brief discussion.

Conformational studies on wheat embryo 5S RNA using nuclease S1 as a probe

1978 ◽  
Vol 56 (5) ◽  
pp. 357-364 ◽  
Author(s):  
Calvin Barber ◽  
J. L. Nichols
Keyword(s):  
Secondary Structure ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Helical Structure ◽  
Fingerprint Analysis ◽  
5S Rna ◽  
Wheat Embryo ◽  
Conformational Studies ◽  
Enzymic Digestion ◽  
Wheat Embryos

Ribosomal 5S 32P-labelled RNA was isolated from imbibing wheat embryos and digested with nuclease S1, a single-strand specific nuclease. The products of enzymic digestion were separated by polyacrylamide gel electrophoresis and identified by fingerprint analysis of their RNase T1 digestion products. The results indicate that the most sensitive portion of the molecule, and hence, the region containing the least helical structure, is close to the 5′-terminus. Similarly, the most resistant portion of the molecule is close to, but does not include, the 3′-terminus. These findings are discussed in relation to proposed models for the secondary structure of 5S RNA.

Physicochemical and chemical studies on wheat embryo ribosomal proteins

1968 ◽  
Vol 46 (4) ◽  
pp. 373-380 ◽  
Author(s):  
Fred H. Wolfe ◽  
Cyril M. Kay
Keyword(s):  
Molecular Weight ◽  
Ribosomal Proteins ◽  
High Speed ◽  
Polyacrylamide Gel Electrophoresis ◽  
Average Molecular Weight ◽  
Optical Rotatory Dispersion ◽  
Random Coil ◽  
Wheat Embryo ◽  
Molecular Weights ◽  
A Value

The physical heterogeneity of unfractionated wheat embryo ribsomal proteins, prepared by the glacial acetic acid method of Waller and Harris, has been investigated in 8 M urea −10−3 M dithio-threitol solutions of low pH (4.5). Sedimentation–diffusion measurements resulted in a weight average molecular weight of 29 000 ± 2 500, with no obvious evidence of heterogeneity. High-speed membrane osmometry was employed to establish the number average molecular weight of this system as 24 500 ± 1 000. The disparity in molecular weight averages suggests some size heterogeneity, and statistical analysis based on the two average molecular weights resulted in a calculated range of molecular weights for wheat embryo ribosomal proteins from 15 000 to 34 000 a.m.u. Charge differences, reflecting presumably primary structure differences, also exist among the members of this class, since about 26 different bands were resolved on polyacrylamide gel electrophoresis. The weight intrinsic viscosity of the ribosomal proteins in 8 M urea solutions was established as 0.273 dl/g, a value considerably larger than most globular proteins, suggesting that a major portion of their polypeptide chains are unfolded in this solvent. This conclusion was substantiated by optical rotatory dispersion measurements on this system, which resulted in a dispersion constant, λc, of 213 m μ, a value typical of that of the random coil. Amino acid and N-terminal analyses are also reported for this system, and comparisons of both chemical and physicochemical parameters are made with ribosomal proteins of other sources.

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 Genetic polymorphism of platelet glycoprotein Ib

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 622-629 ◽  
Author(s):  
M Moroi ◽  
SM Jung ◽  
N Yoshida
Keyword(s):  
Gel Electrophoresis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Glycoprotein ◽  
Bleeding Tendency ◽  
Gene Frequencies ◽  
Specific Staining ◽  
Molecular Weights ◽  
Different Types ◽  
Molecular Weight Difference

Platelet glycoprotein (GP) Ib from 131 healthy Japanese was analyzed using SDS-polyacrylamide gel electrophoresis and specific staining with peroxidase-coupled wheat germ agglutinin after it was transferred to nitrocellulose membranes. Four slightly different species of GPIb were observed and designated as A, B, C, and D for glycoproteins with molecular weights of 168,000, 162,000, 159,000, and 153,000 daltons, respectively. The respective gene frequencies were calculated to be .073, .011, .561, and .355 for A-, B-, C-, D-type GPIb. Portions from each type of GPIb molecule (alpha-chain and glycocalicin) showed heterogeneity with the same molecular weight difference, indicating that the variance would be derived from the polypeptide portion that is exposed to the outer medium. The different types of GPIb were the same with respect to their accessibility to lactoperoxidase, reactivity to lectins, and affinity to TLCK-thrombin. Although Bolin et al reported patients with a bleeding tendency whose platelets have double GPIb bands, here we found that platelets with different GPIb phenotypes showed no significant differences in aggregating activity and platelet retention. Analysis of GPIb phenotype should be important for structural and physiologic studies on GPIb and glycocalicin.

Subcellular distribution and partial characterization of the three major classes of concanavalin A receptors associated with rat brain synaptic junctions

1980 ◽  
Vol 58 (10) ◽  
pp. 941-951 ◽  
Author(s):  
James W. Gurd
Keyword(s):  
Rat Brain ◽  
Concanavalin A ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Structural Heterogeneity ◽  
Molecular Weights ◽  
Receptor Sites ◽  
Synaptic Junctions ◽  
The Individual ◽  
Triton X

Synaptic junctional complexes from rat brain contain three major classes of glycoproteins which react with concanavalin A. They have apparent molecular weights of 110 000 (GP 110) 130 000 (GP 130), and 180 000 (GP 180). They are present in postsynaptic densities but are not found in microsomes, axolemma, synaptic vesicles, or myelin and are present in low concentrations in the Triton X-100 extract obtained during the preparation of synaptic junctions suggesting that they are uniquely localized to the postsynaptic apparatus. Reaction of the individual glycoproteins, partially purified by affinity chromatography on concanavalin A – agarose followed by polyacrylamide gel electrophoresis, showed that GP 130 contained the most receptor sites for concanavalin A per unit of protein followed by GP 180 and GP 110. Of the receptor sites for concanavalin A, 60–70% were subject to hydrolysis by endoglycosidase H indicating that the lectin reacts primarily with polymannose asparagine linked oligosaccharides. Each of the glycoproteins also reacted to varying degrees with the lectins from Lotus tetragonolobus (specific for α-L-fucose), wheat germ (N′-acetyl-D-glucosamine and (or) sialic acid), and lentils (mannose, N′-acetyl-D-glucosamine). Chromatography of 125I-labelled concanavalin A positive glycoproteins on wheat germ Sepharose resolved GP 110 and GP 180 into wheat germ positive and negative components indicating the presence of some structural heterogeneity within these molecular weight classes.

scholarly journals Accelerated breakdown of reticulocyte protein formed under conditions of amino acid depletion

1978 ◽  
Vol 176 (1) ◽  
pp. 151-158 ◽  
Author(s):  
C S Chandler ◽  
F J Ballard
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Stringent Response ◽  
Molecular Weights ◽  
Protein Inhibition ◽  
Deficient Medium ◽  
Amino Acid Depletion ◽  
Rabbit Reticulocytes

1. Labile protein is formed when rat or rabbit reticulocytes are incubated in medium deficient in individual amino acids, especially histidine, valine or alanine. The fraction of unstable protein is increased to about 35% of the total protein synthesized when the histidinyl-tRNA-charging inhibitor, histidinol, is added to histidine-deficient media. 2. The molecular weights of the labile proteins measured by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of urea are less than haemoglobin and probably represent prematurely terminated haemoglobin chains. 3. Although protein synthesis is always lower under conditions that produce labile protein, inhibition of protein synthesis by fluoride or cycloheximide does not give an effect similar to amino acid depletion. 4. The synthesis of protein in deficient medium does not alter the degradation rate of pre-existing protein in reticulocytes and is thus unrelated to the stringent response in bacteria. 5. We propose that amino acid-deficient medium leads to a decreased charging of the appropriate tRNA, a concomitant decrease in protein synthesis and the degradation of nascent peptides.

Wheat embryo ribonucleates XI. Conserved mRNA in dry wheat embryos and its relation to protein synthesis during early imbibition

1978 ◽  
Vol 56 (6) ◽  
pp. 365-369 ◽  
Author(s):  
A. C. Cuming ◽  
B. G. Lane
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Free System ◽  
Wheat Embryo ◽  
Free Translation ◽  
Wheat Embryos ◽  
The Many

It has been found that bulk poiy(A)-rich RNA from dry wheat embryos is broadly hetero-disperse when examined by polyacrylamide gel electrophoresis. The poly(A)-rich RNA from dry wheat embryos has been translated in a cell-free protein-synthesizing system from the same commerically supplied, roller-milled wheat embryos. Compatible with the electrophoretic heterodispersity observed for poly(A)-rich RNA, the radioactive products of its cell-free translation, when examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, have mobilities that are broadly coincident with the many dye-stained (nonradioactive) proteins present in wheat extracts. With due allowance for the limitations of the cell-free system, which is known to translate, selectively, lower molecular-weight species of mRNA, it has been concluded that the conserved poly(A)-rich mRNA in dry wheat embryos probably has the translational capacity required to account for the highly eclectic protein synthesis that we have observed during early (40-min) imbibition of viable wheat embryos.

Wheat embryo ribonucleates. VIII. The presence of 7-methylguanosine 'cap structures' in the RNA of imbibing wheat embryos

1977 ◽  
Vol 55 (8) ◽  
pp. 819-824 ◽  
Author(s):  
M. S. Saini ◽  
B. G. Lane
Keyword(s):  
Ribosomal Rna ◽  
Messenger Rna ◽  
Large Fraction ◽  
Deae Cellulose ◽  
Careful Analysis ◽  
Significant Fraction ◽  
Unbound Fraction ◽  
Wheat Embryo ◽  
Wheat Embryos ◽  
The Subject

1. By imbibing wheat embryos in media that contain methyl-labelled methionine, it is possible to label both terminal and nonterminal 7-methylguanosine constituents in NaCl-insoluble (2.5 M, 0 °C) RNA (iRNA).2. Most of the 7-[Me-14C]methylguanosine in wheat embryo i[Me-14C]RNA is present in nonterminal positions of polynucleotide chains, probably in ribosomal RNA.3. By passage through a column of oligo-dT-cellulose, it is possible to show that most of the 7-[Me-3H]methylguanosine in a 'bound' fraction of i[Me-3H]RNA from imbibing wheat embryos is present in terminal 'cap' structures, probably in messenger RNA.4. Although most of the 7-[Me-3H]methylguanosine in the 'unbound' (to oligo-dT-cellulose) fraction of i[Me-3H]RNA was present in nonterminal positions, there was also a highly significant fraction of 7-[Me-3H]methylguanosine in terminal 'cap' structures. Although it will be a subject of continued investigation, possible reasons why a large fraction of the total 7-[Me-3H]-methylguanosine was present in the 'unbound' fraction, in this present study, are a subject of discussion.5. Careful analysis failed to reveal the presence of any N6,O2′-di[Me-3H]methyladenosine in the 'unbound' fraction of i[Me-3H]RNA.6. Factors that might influence the binding of 'cap' oligonucleotides to DEAE-cellulose are the subject of a brief discussion.

scholarly journals Genetic polymorphism of platelet glycoprotein Ib

Blood ◽  
1984 ◽  
Vol 64 (3) ◽  
pp. 622-629 ◽  
Author(s):  
M Moroi ◽  
SM Jung ◽  
N Yoshida
Keyword(s):  
Gel Electrophoresis ◽  
Wheat Germ ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Glycoprotein ◽  
Bleeding Tendency ◽  
Gene Frequencies ◽  
Specific Staining ◽  
Molecular Weights ◽  
Different Types ◽  
Molecular Weight Difference

Abstract Platelet glycoprotein (GP) Ib from 131 healthy Japanese was analyzed using SDS-polyacrylamide gel electrophoresis and specific staining with peroxidase-coupled wheat germ agglutinin after it was transferred to nitrocellulose membranes. Four slightly different species of GPIb were observed and designated as A, B, C, and D for glycoproteins with molecular weights of 168,000, 162,000, 159,000, and 153,000 daltons, respectively. The respective gene frequencies were calculated to be .073, .011, .561, and .355 for A-, B-, C-, D-type GPIb. Portions from each type of GPIb molecule (alpha-chain and glycocalicin) showed heterogeneity with the same molecular weight difference, indicating that the variance would be derived from the polypeptide portion that is exposed to the outer medium. The different types of GPIb were the same with respect to their accessibility to lactoperoxidase, reactivity to lectins, and affinity to TLCK-thrombin. Although Bolin et al reported patients with a bleeding tendency whose platelets have double GPIb bands, here we found that platelets with different GPIb phenotypes showed no significant differences in aggregating activity and platelet retention. Analysis of GPIb phenotype should be important for structural and physiologic studies on GPIb and glycocalicin.

Wheat-Embryo Ribonucleates. I. Subcellular Localization of a Satellite Polyribonucleotide

1973 ◽  
Vol 51 (5) ◽  
pp. 606-612 ◽  
Author(s):  
A. A. Azad ◽  
B. G. Lane
Keyword(s):  
Microsomal Fraction ◽  
Large Subunit ◽  
Satellite Rna ◽  
Selective Precipitation ◽  
Ribosomal Subunits ◽  
Wheat Embryo ◽  
Wheat Embryos ◽  
The Subject ◽  
Rna Complex ◽  
Electrophoretic Component

(1) When wheat embryos are extracted with aqueous phenol and the aqueous phase is made 2.5 M with respect to NaCl at 0 °C, there is selective precipitation of about 80% of the total RNA. The 18 S and 26 S RNA species from the wheat-embryo ribosomes comprise a preponderant mass fraction (ca. 80%) of this NaCl-insoluble RNA (iRNA). A small amount of a rapidly migrating electrophoretic component (iRMEC) can be released by aqueous denaturation of wheat-embryo NaCl-insoluble RNA and because it is specifically complexed with 26 S RNA, the iRMEC component has been termed a "satellite" of 26 S RNA.(2) The wheat-embryo satellite RNA has been shown to be present in the microsomal fraction recovered from cell-free homogenates of wheat embryos.(3) The wheat-embryo satellite RNA has been shown to be differentially localized in the large subunit of wheat-embryo ribosomes where it presumably exists as part of the same intermolecular 26 S RNA complex that can be isolated by directly extracting the whole embryos with aqueous phenol.(4) During preparation of the ribosomal subunits, there is substantial degradation of the component ribonucleates and the nature of this degradation is the subject of a brief discussion.

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