Rabbit globin mRNA species containing poly(A) segments of different lengths were prepared by partial phosphorolysis of mRNA with Escherichia coli polynucleotide phosphorylase. By varying the salt concentration and the time of incubation of the phosphorolysis mixture, as well as performing oligo(dT)-cellulose chromatography at 22 "C and at 4 "C, globin mRNA preparations containing poly(A) segments of approximately 122, 95, 68, 39, 32,21, and 16 adenylate residues were obtained. It was found that the functional stability of the mRNA species containing 32 or more adenylate residues after injection into Xenopus luevis oocytes equaled that of the native globin mRNA. On the other hand, the functional stability of mRNA containing an average number of 21 adenylate residues was about 30% of the native mRNA, while that of mRNA containing 16 adenylate residues was as low as poly(A)-free globin mRNA.The existence of poly(A)-rich sequences at the 3'-end of most eukaryotic mRNAs is well established (for reviews see [l -31). The biological role of the poly(A) segment was the subject of many recent studies. Inhibition of post-transcriptional adenylation by the adenosine analogue cordycepin strongly reduces the flow of mRNA from the nucleus to the cytoplasm. This finding forms the basis for the suggestion that nuclear adenylation is an essential step in the biogenesis of mRNA or in its transport to the cytoplasm [4,5].Other findings, such as shortening of the poly(A) segment during the aging of mRNA [6,7] cytoplasmic adenylation [8,9], and the presence of a poly(A) segment in a viral message which is synthesized in the cytoplasm [lo], suggest an additional role for the poly(A) segment outside of the nucleus. Results reported from this and other laboratories have shown that the poly(A) segment of globin mRNA is not essential for its translation in cell-free systems [l 1 -161. However, preincubation in cell-free extracts decreases the template activity of poly(A)-free mRNA more than that of the native mRNA [16]. We have also reported that rabbit globin poly(A)-free mRNA is translated as efficiently as native mRNA during the Enzyme. Nucteosidediphosphate : polynucleotide nucleotidyltransferase (polynucleotide phosphorylase) (EC 2.7.7.8).first 45-min period of incubation in a Krebs-I1 ascites cell-free systems, but upon longer periods of incubation the rate of protein synthesis decreases more rapidly with the poly(A)-free mRNA than with the native mRNA [Ill. To overcome the time limitation of the cell-free system (which is active for only 90 min) we have used Xenopus luevis oocytes, which permit mRNA translation for some days. A pronounced difference between the functional stability of poly(A)-free and native globin mRNA was observed : whereas efficiency of translation of poly(A)-free mRNA in the oocytes is the same as that of the native globin mRNA during the first few hours after injection, 20-48 h later the efficiency of translation drops to about 10% of that of native globin mRNA [12]. Recently we have found that the drop in tem...

The translations of native messenger RNA for rabbit globin and that of poly(A)-free globin messenger RNA have been compared after injection into XenopUs oocytes. The initial rate of translation of poly-(A)-free miINA is close to that found with intact mRNA.However, at longer incubation periods, the rate of globin synthesis with poly(A)-free mRNA is considerably lower than with native mRNA. Similar differences in the template activity of the two mRNA preparations were found with a cell-free extract of Krebs II ascites tumor. It is concluded that the presence of the 3' poly(A)-rich sequence in mRNA is required to ensure high functional stability.

Rabbit globin poly(A)free and native mRNA preparations were microinjected into Xenopus oocytes. The amount of globin message remaining after incubation of injected oocytes was determined by molecular A specific method has been developed for removal of the 3'-poly(A) segment from mRNA. The method is based on synchronous processive phosphorolysis of mRNA using molar excess of Escherichia colh polynucleotide phosphorylase at 00 in the presence of salt (1). Rabbit globin poly(A)-free mRNA, obtained by this method, can still be translated in Krebs II ascites tumor cell-free extracts. It was also observed that, after longer periods of incubation, the activity of the poly(A)-free mRNA had leveled off earlier than that of native mRNA(l). A more pronounced difference was found upon microinjection of the mRNA preparation into Xenopus oocytes. This in vivo system is more efficient than the in vitro cell-free system and the translation process can be followed for several days. It was found under these conditions that the frog oocytes, poly(A)-free mRNA is translated into globin chains for a much shorter period of time than native poly(A)-containing mRNA (2). However, the means by which translation of poly(A)-free mRNA is prevented are not known. It is possible that removal of the poly(A) segment might somehow reduce the stability of the mRNA and lead to its degradation. Alternatively, some other process may inactivate poly(A)-free mRNA and interfere with its translation (1, 2). To answer this question, we have estimated the amount of message remaining in oocytes after microinjection of poly(A)-free or native mRNA preparations.MATERIALS AND METHODS Solutions Used. Phenol-CHCl3 mixture: a mixture of 100 g of phenol, 14 ml of cresol, and 0.5 g of 8-hydroxyquinoline is equilibrated several times with buffer A until the correct pH (8)

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As shown previously by several authors, cycloheximide inhibits progesterone‐induced maturation. However, normal maturation can be obtained, in Xenopus, after a 5 h treatment with cycloheximide (10–20 μ/ml), followed by extensive washing. Under these conditions, protein synthesis is still 50% inhibited. When X.laevis oocytes are continuously treated with a progesterone and cycloheximide mixture, they undergo a degenerative process which has been termed ‘pseudomaturation’: the germinal vesicle membrane breaks down, but the chromosomes do not condense and the nucleoli do not disappear completely. Progesterone‐induced maturation was not arrested by fusidic acid in Rana pipiens, even after micro‐injection. Actinomycin D (10–20 μg/ml) speeds up maturation in both R. pipiens and X. laevis. Microinjection of α‐amanitin into control R. pipiens oocytes does induce maturation in a few cases. It is concluded that one of the effects of progesterone might be a repression of RNA synthesis. Successive treatments with cycloheximide and actinomycin D failed to induce maturation, but often produced ‘pseudomaturation’ in X. laevis. Injection of a cytoplasmic extract (centrifuged homogenate) from X. laevis eggs which have undergone maturation into recipient oocytes of the same species induces ‘pseudomaturation’ and strongly inhibits protein synthesis. If the membranes which surround the egg are eliminated before homogenization, true maturation is obtained. The membranous material apparently releases factors which exert a negative effect on maturation and protein synthesis. Homogenates from eggs which have not been treated with progesterone do not, after injection, induce ‘pseudomaturation’ and have little effect on protein synthesis. In contrast with the findings of Ecker and Smith (1971)11 on R. pipiens, protein synthesis in X. laevis, after a short stimulation, drops considerably (50%) during progesterone‐induced maturation.