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.
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 alpha-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 alpha-tubulin mRNA and total polyadenylated mRNA are largely unaffected when the rates of tubulin and total protein synthesis vary dramatically. Thus, differential regulation of alpha-tubulin mRNA translation initiation does not contribute to the greater than 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.
Calzone
F J
FJ
Department of Biology, University of Rochester, NY 14627.
Callahan
R
R
Gorovsky
M A
MA
D000180:Acrylic Resins; D000645:Ammonium Sulfate; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D002499:Centrifugation, Density Gradient; D002923:Cilia / Q000502:physiology*; D005029:Ethylenediamines; D015336:Molecular Probe Techniques / Q000379:methods; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D011132:Polyribosomes / Q000032:analysis / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D013768:Tetrahymena / Q000254:growth & development* / Q000502:physiology; D014404:Tubulin / Q000096:biosynthesis / Q000378:metabolism*
D000180:Acrylic Resins; D005029:Ethylenediamines; D012333:RNA, Messenger; D014404:Tubulin; C016680:polyacrylamide gels; C005798:N,N,N',N'-tetramethylethylenediamine; C031276:ammonium peroxydisulfate; D011062:Polynucleotide Adenylyltransferase; D000645:Ammonium Sulfate