Erik De Herdt

Membrane associated poly (A)-containing ribonucleoproteins in Artemia Salina gastrula embryos

The embryonic development of Artemia salina is often interrupted by a period of dormancy at the gastrula stage. Although no endogeneous protein synthesis could be detected in these encysted embryos [ 11, the presence of stored mRNA has been demonstrated [2-4]. Messenger activity is associated with both poly(A)+-RNA and poly(A)--RNA extracted from the membrane fraction but only with the poly(A)-RNA of the free cytoplasmic ribonucleoproteins [3]. Poly(A)*-RNA of the cytoplasmic RNP is complexed with a translational inhibitor RNA, and messenger activity is restored if the inhibitor RNA is dissociated from the mRNA by the use of EDTA during the fractionation procedure [ 51. We have shown by sucrose gradient centrifugation that free cytoplasmic poly(A)+RNPs exhibited a heterogeneous distribution consisting of several discrete peaks ranging from 20-120 S [4]. Approximately 50% of the RNPs sedimented faster than 50 S and were complexed with ribosomes or ribosomal subunits. The presence of EDTA converted all these poly(A)*-RNPs into free 20-40 S particles. We demonstrate here that the poly(A)‘-RNPs of the membrane fraction exhibit a different size distribution on sucrose gradients as compared with the

Characterization of protein components of poly(A)-containing messenger ribonucleoproteins from cryptobiotic gastrulae of Artemia salina.

Conditions for the isolation of intact poly(A)+mRNP from cryptobiotic gastrulae ofA. salina are described. In the presence of Mg2+ ions nucleolytic cleavage occurs in vitro in the vicinity of the 3′-poly(A) segment of mRNP during the isolation procedure. The resulting two parts of poly(A)+mRNP complex are separated by thermal elution from oligo(dT)-cellulose affinity column. Analysis by SDS-gel electrophoresis of protein components associated with intact poly(A)+mRNP has revealed the existence of 20–30 S RNP complex containing five major proteins with Mr 68,000, 53,000, 50,000, 45,000 and 38,000, respectively, but completely lacking the poly(A)-specific Mr 76,000 protein.

Affinity chromatography of poly(adenylic acid)-containing ribonucleoproteins on oligothymidylic acid-cellulose.

Binding of poly(A)-containing RNP to oligo(dT)-cellulose has been investigated as a function of mono- and divalent ion concentration. 80-90% binding was obtained either in high (500 mM) or in moderate NaCl concentrations in the presence of 5 mM MgCl2. At 40 mM NaCl and 5 mM MgCl2 poly(A)+-RNP exhibit approximately the same stability as poly(A)+-RNA in binding to oligo(dT)-cellulose with a melting temperature of 41 and 45 degrees C, respectively, indicating that the protein moiety has no effect on the ribonucleoprotein binding in these conditions. Differences were observed in the elution of poly(A)+-RNA and poly(A)+-RNP from oligo(dT)-cellulose in buffer without salts. Poly(A)+-RNA was completely removed at 4 degrees C whereas the melting temperature of poly(A)+-RNP was only decreased to 34 degrees C. The isolation of poly(A)+-RNP by thermal elution from oligo(dT)-cellulose is described.