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Direct measurement of tubulin and bulk message distributions on polysomes of growing, starved and deciliated Tetrahymena using RNA gel blots of sucrose gradients containing acrylamide

1988; Oxford University Press; Volume: 16; Issue: 20 Linguagem: Inglês

10.1093/nar/16.20.9597

1362-4962

Frank J. Calzone, Rosemary Callahan, Martin A. Gorovsky,

Photosynthetic Processes and Mechanisms

A method was developed using sucrose gradients containing acrylamide which greatly simplifies the measurement of the polysomal distribution of messages.After centrifugation, the acrylamide was polymerized, forming a "polysome gel".RNA gel blots of polysome gels were used to determine the polysomal distributions of a-tubulin and total polyadenylated mRNA in growing, starved (nongrowing) and starved-deciliated Tetrahymena and the number of messages loaded onto polysomes was calculated.These measurements indicated that the translational efficiencies of a-tubulin mRNA and total polyadenylated mRNA are largely unaffected when the rates of tubulin and total protein synthesis vary dramatically.Thus, differential regulation of a- tubulin mRNA translation initiation does not contribute to the >100-fold induction of tubulin synthesis observed during cilia regeneration and in growing cells.The major translation-level process regulating tubulin synthesis in Tetrahymena appears to be a change in message loading mediated by a non-specific message recruitment or unmasking factor. INTRODUCIIONThe efficiency of mRNA translation (polypeptides produced per mRNA per unit time) and the efficiency of mRNA loading onto polysomes are two important factors determining the level of expression of genes.A common approach to evaluating these parameters for a specific message employs sucrose gradient centrifugation to measure the size of polysomes translating the message and the polysomal and nonpolysomal distribution of the message.The analysis of material separated by sucrose gradient centrifugation is tedious.Specific messages or proteins must be assayed in each gradient fraction requiring many parallel determinations to be performed for each experiment.We have developed a technique which greatly simplifies the assay of message distributions in sucrose gradients.Sucrose gradients containing acrylamide can be used to separate polysomes.After centrifugation the acrylamide can be polymerized and RNA (or protein) can be blotted from the "polysome gel" to filters by conventional techniques.The resulting polysome gel blots can be probed with gene (or antibody) probes to locate messages in the image of the sucrose gradient transferred to the filter.We have employed polysome gels to examine the efficiency of tubulin mRNA translation in starved (nongrowing), growing and starved-deciliated Tetrahymena.After deciliation starved Tetrahvrna regenerate cilia and tubulin synthesis is increased more than 100-fold while the overall rate of protein synthesis increases about 10-fold (1, 2).In starved Tetrahymena only 4% of