Isolation of RNA with Properties of Messenger RNA from Cerebral Polyribosomes

Zomzely, Claire; Roberts, Sidney; Peache, Susan

doi:10.1073/pnas.67.2.644

Cited by 25 publications

(8 citation statements)

References 39 publications

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“…4). Preparations of ribosomal wash factors which contain initiation, as well as a variety of other factors, have been shown to be necessary for the translation of brain mRNAs (Zomzely et al, 1970; Zomzely-Neurath et a]. , 1972; Gilbert, 1974).…”

Section: Discussionmentioning

confidence: 99%

Homologous Mammalian Brain Cell Lysate System for the Initiation and Translation of Exogenous mRNAs

Gilbert¹,

Mattick²

1981

Journal of Neurochemistry

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The rate of protein synthesis in mammalian brain tissue is affected by a variety of physiological conditions, both natural and induced. The process of initiation may be involved in some of the observed changes, although as yet the actual rates of initiation of natural mRNAs have not been directly measured in these circumstances. One approach to studying the regulation of protein synthesis in brain tissue would be to utilize a homologous cell‐free system to examine in vitro the translation of various added mRNAs. The present report describes a micrococcal nuclease‐treated cell‐free lysate system derived from fetal mouse brain tissue which is capable of actively initiating and translating exogenously added mRNA. Sodium dodecyl sulfate‐polyacrylamide slab gel electrophoretic analysis of the specific protein products of the reaction mixture allowed a qualitative and quantitative assessment of the translational process under a variety of experimental conditions. Optimal conditions for mRNA‐dependent protein synthesis were the following: 30°C incubation temperature; 80–100 mM‐KCl; 2.1 mM‐Mg2+; 50 μM‐spermhe; and 10 μg/ml poly A(+) mRNA. Incorporation of L‐[35S]methionine into proteins required ATP, GTP, and an energy regenerating system. The addition of saturating amounts of a homologous “initiation factors” fraction stimulated incorporation twofold during the first 20 min of incubation, while the patterns of inhibition observed upon the addition of 5 × 10‐5 M‐aurin tricarboxylic acid at various periods during incubation demonstrated the occurrence of multiple rounds of initiation.

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Section: Discussionmentioning

confidence: 99%

Homologous Mammalian Brain Cell Lysate System for the Initiation and Translation of Exogenous mRNAs

Gilbert¹,

Mattick²

1981

Journal of Neurochemistry

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“…An RNA fraction containing mRNA-like material (DNA-like base composition, high level of DNA-RNA hybridization, stimulation of amino-acid incorporation in vitro), prepared from cerebral polysomes (47), was used in experiments designed to investigate de novo synthesis of specific proteins by this cerebral cell-free system. S100 protein was used as the marker for evidence of in vitro synthesis of a specific protein programmed by an isolated mRNA fraction.…”

Section: Resultsmentioning

confidence: 99%

“…Isolation of Polysome-Derived mRNA. RNA, released by EDTA treatment of total polyribosomes from cerebral white matter and whole brain of rats, was prepared as described by Zomzely, Roberts, and Peache (47). This polysome-derived RNA was shown, by indirect criteria, to have properties expected of mRNA.…”

mentioning

confidence: 99%

Synthesis of a Brain-Specific Protein (S100 Protein) in a Homologous Cell-Free System Programmed with Cerebral Polysomal Messenger RNA

Zomzely-Neurath

York

Moore

1972

Proc. Natl. Acad. Sci. U.S.A.

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Polyribosomes, carrying nascent polypeptide chains, were prepared from whole brain, cortex, and hindbrain-medullary white matter of young adult rats. In a homologous cell-free system, a brain-specific protein (S100 protein) was identified in the mixture of polypeptides released from the polyribosomes during incubation for 1 hr at 37°. De novo synthesis of the S100 protein was achieved in a reconstituted cerebral cell-free system containing polysome-derived mRNA and 40S + 60S subunits. The radioactively labeled S100 protein synthesized in vitro was identified by precipitation with antibody to S100 after addition of purified S100 as a carrier, and migration of the solubilized precipitate on acrylamide gels in the presence of sodium dodecyl sulfate. In vitro synthesis of the S100 protein did not occur in analogous cell-free systems derived from hepatic tissue or in a heterologous system containing liver polyribosomes and cerebral enzymes.One of the active areas of research on mammalian protein biosynthesis has been the identification of products formed during cell-free incorporation of amino acids by polyribosomal preparations. The demonstration of active incorporation into globin chains by particles from reticulocytes was the first example of clear-cut evidence for an identifiable product formed in vitro (1-3). Some of the more recent and numerous examples include the demonstration of active incorporation into serum proteins and nonserum liver proteins by particulate preparations from hepatic tissue (4-11), into myosin by polyribosomes from embryonic-chick muscle (12) and myoglobin by polysomes from duck skeletal muscle (13), into heavy and light chains of antibodies by polysomes from rabbit lymph nodes (14, 15) and ovalbumin by chick-oviduct polysomes (16), and into B-lactoglobulin by particles from ewe mammary gland (17).Although cell-free systems dervied from brain are highly active in amino-acid incorporation (18-21), identification of products formed in vitro is difficult because of the complexity and heterogeneity of cerebral tissue. Nevertheless, Rubin and Stenzel (22) reported the in vitro synthesis of a soluble acidic protein (S100 protein) by a ribosomal system from rabbit cortical gray matter. Mahler and Brown (21) later reported that in a polysomal system from rat brain, about 33% of the counts released into the soluble portion during cell-free protein synthesis were found among several acidic proteins, but identification of a specific protein was not attempted in their studies. The isolation, purification, and characterization of two brain-specific low molecular weight proteins by Moore et al., as well as preparation of antisera to these proteins, has made possible a study of the in vitro synthesis of specific brain proteins by cerebral polyribosomes. The S100 protein (23) is primarily of glial origin (24-26), and represents 0.2% of the total soluble protein of brain. The other purified protein (14-3-2) is localized primarily in neurons (27). The function of these proteins is unknown, but t...

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“…The type of RNA change observed in these experiments can be accounted for by a shift in the pattern of RNA synthesis from one predominant in ribosomal RNA content to one containing largely nuclear heterodisperse and messenger RNA. The interpretation of the data in these terms is provided by the base-composition measurements of brain RNA reported by Zomzely, Roberts, and Peache (14). According to these authors, the U/C ratios are 0.78 for ribosomal RNA, 1.33 for "nuclear hybridizable RNA", and 1.54 for the RNA associated with polysomes.…”

mentioning

confidence: 97%

Dibutyryl Adenosine Cyclic 3′:5′-Monophosphate Effects on Goldfish Behavior and Brain RNA Metabolism

Shashoua¹

1971

Proc. Natl. Acad. Sci. U.S.A.

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Intraventricular administration of dibutyryl adenosine cyclic 3':5'-monophosphate into goldfish brains produced hyperactive animals. A study of the effects of the drug (25-50 mg/kg) on the incorporation of [5-'Hlorotic acid, as a precursor of labeled uridine and cytidine, into newly synthesized RNA showed the formation of an RNA with a uridine to cytidine ratio 20-50% higher than that of the control. In double-labeling experiments with uridine as the labeled precursor, the synthesis of a nuclear RNA fraction (not produced in the absence of drug) was demonstrated. Some of this RNA was found to migrate into the cytoplasmic fraction and to become associated-with polysomes. The results suggest that cyclic AMP might function as a "metabolic demand signal" for eliciting new RNA synthesis in goldfish brain.Adenosine cyclic 3':5'-monophosphate (cAMP) has been implicated as a mediator of hormonal responses in many cell functions. Sutherland et al. (1, 2) have proposed the general concept that cAMP acts as the "second messenger" inside cells. According to this postulate, the external receptor molecules are coupled with adenylate cyclase molecules situated on the internal surface of the cell membrane. Inputs, hormonal or otherwise, at the external receptors would promote some type of conformational change to activate the synthesis of cAMP by adenylate cyclase. By this means, environmental inputs would be transduced by the surface receptors into an increased intracellular concentration of cAMP. The cAMP molecules within the cell then participate in various functions.A number of observations have suggested that cAMP also plays a key role in brain metabolism. Sutherland, Rall, and Menon (3) found that adenylate cyclase is widely distributed in brain tissue; the concentration was highest in the cortex and the lowest in the spinal cord of rat brain. Kakiuchi and Rall (4) showed that tissue slices from brain can be stimulated to synthesize cAMP by catecholamines, particularly norepinephrine. In addition, electrical stimulation of nervous-tissue slices (5) or of intact superior cervical sympathetic ganglia (6) produces a 10-fold and a 5-fold increase in the concentration of cAMP, respectively. Electrophoretic administrations of cAMP to cerebellar Purkinje cells causes a rapid decrease in the firing rate of the neurons (7), suggesting a post synaptic role for the molecules.In a previous paper (8) RNA changes in the brain of goldfish were correlated with the acquisition and storage of new behavioral patterns. It was suggested (8) that the RNA changes might result from a "biochemical demand signal" that was characteristic of the response of the nervous system to the acquisition of new behavioral patterns. In the present paper, the possibility that cAMP may function as such a "biochemi-2835 cal demand signal" was examined. Two experimental methods were used in-the study. In the first series of experiments, intracerebral injections of N6,021-dibutyryl adenosine cyclic havior was studied by injection of 10-IA aliquots of an aqu...

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Isolation of RNA with Properties of Messenger RNA from Cerebral Polyribosomes

Cited by 25 publications

References 39 publications

Homologous Mammalian Brain Cell Lysate System for the Initiation and Translation of Exogenous mRNAs

Homologous Mammalian Brain Cell Lysate System for the Initiation and Translation of Exogenous mRNAs

Synthesis of a Brain-Specific Protein (S100 Protein) in a Homologous Cell-Free System Programmed with Cerebral Polysomal Messenger RNA

Dibutyryl Adenosine Cyclic 3′:5′-Monophosphate Effects on Goldfish Behavior and Brain RNA Metabolism

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