Partial purification of the alpha-tubulin and beta-tubulin messenger RNAs from rat brain.
Poly(A)-containing RNA from frozen adult rat brain were fractionated by centrifugation in a formamide/sucrose gradient. Individual fractions were used to program protein synthesis in vitro in a reticulocyte lysate. The cell-free translation products were analyzed by two-dimensional electrophoresis in polyacrylamide slab gels. We observed a heterodispersion of the mRNA translation activity coding for the beta-tubulin subunit which contrasts with a relatively homogeneous distribution of the alpha-tubulin subunit mRNA. These last mRNA species are present in a peak which sediments near the 18-S region of the gradient whereas the beta-tubulin mRNA activity is predominant in the fractions corresponding to the heaviest mRNA species. When these heaviest RNAs were separated again by centrifugation in a second formamide/sucrose gradient, a poly(A)-rich RNA population was obtained that was enriched in RNA for programming the beta-tubulin subunit. Analysis of the products whose synthesis in vitro was directed by this mRNA population revealed that beta tubulin was the main protein formed, the ratio beta/alpha being more than tenfold greater than in the products translated in vitro using total poly(A)-rich RNA.
D000818:Animals; D001923:Brain Chemistry; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D011061:Poly A / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D014404:Tubulin / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D014404:Tubulin; D011061:Poly A; D012313:RNA