Synthesis of the Brain‐Specific S‐100 Protein in a Cell‐Free System from Wheat Embryo Programmed with Poly(A)‐Containing RNA from Rabbit Brain

Abstract

Polyadenylated polysonial RNA was prepared from rabbit cerebral hemispheres using phenol extraction and chromatography on oligo(dT)‐cellulose. This RNA directed the synthesis of the brain‐specific S‐100 protein in cell‐free extracts front wheat embryo. S‐100 protein was absent from the products of endogenous incorporation and from a reaction programmed with kidney mRNA. These results suggest that S‐100 protein mRNA contains a poly(adenylic acid) sequence and rule out the necessity of a brain‐specific factor for translation of S‐100 protein mRNA.

In Vitro Synthesis of Polypeptides of Moderately Large Size by Poly(A)‐Containing Messenger RNA from Postmortem Human Brain and Mouse Brain

“…The mRNA was active in a wheat germ protein-synthesizing system; however, few products were detected with masses above 70,000 daltons. This may be attributed, in part, to limitations of the wheat germ system, such as endogenous ribonuclease activity (Scheele and Blackburn, 1979), premature termination of polypeptide chains (Benveniste et al, 1976;Tse and Taylor, 1977), the presence of endogenous inhibitor (Zagorski, 1978), and the lower specific activity of brain mRNA in a wheat germ system com- pared with mRNA from a variety of other sources (Mahoney et al, 1976;Giudice and Chaiken, 1979;Matthees and Campagnoni, 1980). Mumford et al (1981) recently demonstrated that although wheat germ and rabbit reticulocyte lysates contain proteolytic activity, the wheat germ lysate had considerably higher levels of aminopeptidase and elastase activities than did the reticulocyte lysate.…”

“…During the present studies it was established that the presence of a protease inhibitor, aprotinin, during synthesis in the reticulocyte lysate improved neither the specific activity of added brain mRNA nor the size distribution of the synthesized products. With the methodology currently available the wheat germ system appears best suited for the in vitro synthesis of lower molecular weight proteins by brain messenger RNA, such as S-100 protein (Mahoney et al, 1976), brain tubulin and actin (Hunter and Garrels, 1977;Bryan et al, 1978, Marotta et al, 1979Cleveland et al, 1979;Portier et al, 1980), myelin basic protein (Matthees and Campagnoni, 1980), and glial fibrillary acidic protein (Beguin et al, 1980;Bigbee and Eng, 1982).…”

“…Most investigations have utilized mRNA prepared from purified polysomes or from total cytoplasmic RNA and translated in a wheat germ homogenate or a reticulocyte lysate. The various methodologies have proved essential for investigating the in vitro synthesis of brain proteins such as S-100 protein (Mahoney et al, 1976), brain tubulin (Bryan et al, 1978;Marotta et al, 1979;Cleveland et al, 1979;Portier et al, 1980;Gozes et al, 1980), brain actin (Hunter and Garrels, 1977;Marotta et al, 1979), myelin basic protein (Mathers and Campagnoni, 1980), and glial fibrillary acidic protein (Beguin et al, 1980;Bigbee and En&, 1982).…”

Synthesis of S100 Protein on Free and Membrane‐Bound Polysomes of the Rabbit Brain

“…The relative synthesis of SlOO protein by free and membrane-bound polysomes of the cerebral hemispheres and cerebellum was investigated. Since SlOO protein is a minor protein, constituting only 0.1-0.2% of the total nascent proteins synthesized in the mammalian brain (Mahony et al, 1976), quantitation of this protein in the cell-free translation products of isolated polysomes was accomplished by direct immunoprecipitation using antisera specific for SlOO protein.…”

“…The cell-free synthesis of SlOO protein has been reported in reconstituted homologous systems derived from rat brain and in heterologous systems programmed with mRNA or polysomes from the brains of several mammalian species (Zomzely-Neurath e t al., 1972;Amaldi et al, 1973;Mahony et al, 1976;Marks et al, 1980b;Mahony and Brown, 1980;reviewed by Zomzely-Neurath and Walker, 1980). In a previous study we have analyzed mRNA coding for S 100 protein during the development of the rabbit brain by means of in vitro translation in a wheat embryo extract (Mahony and Brown, 1980).…”