Abstract
Polysomal RNA was isolated from pea epicotyls treated with (2,4-dichlorophenoxy)acetic acid, and fractionated on oligo(dT)-CELLULOSE TO YIELD POLY(A)-containing RNA. This RNA fraction was translated in a wheat embryo cell-free system and found to have more than 90% of the messenger activity in total polysomal RNA. Immunoprecipitation of the translation product by monospecific antibodies to pea cellulases (beta-1,4-glucan 4-glucanohydrolase, EC 3.2.1.4) indicated that cellulase was synthesized in this system. The immunoprecipitate co-migrated with the buffer-soluble cellulase component in sodium dodecyl sulfate-gel electrophoresis. Buffer-insoluble cellulase was not detected in the in vitro translation products. Fractionation of mRNA from membrane-bound and free polysomes and their subsequent translation indicated preferential synthesis of buffer-soluble cellulase on membrane-bound polysomes. With the above techniques for assaying buffer-soluble cellulase mRNA, a 10-fold increase in the level of this messenger per tissue segment was observed within 48 hours following (2,4-dichlorophenoxy)acetic acid treatment. There was no evidence for pre-existing untranslated message for cellulase in control tissues. Since there was no delay in the appearance of mRNA for cellulase, compared to a 24-hour lag in the increase of cellulase activity, it is suggested that translational as well as transcriptional controls are exerted on the biosynthesis of cellulase in vivo. Analysis of the rates of peptide chain initiation and elongation, both in vivo and in vitro, indicated that peptide chain elongation may be rate-limiting during the lag phase of cellulase development.
Highlights
Indicated that cellulase was synthesized in this sytem
With the above techniques for assaying buffer-soluble cellulase mRNA, a IO-fold increase in the level of this messenger per tissue segment was observed within 48 hours following (2,4-dichlorophenoxy)acetic acid treatment
Since there was no delay in the appearance of mRNA for cellulase, compared to a 24-hour lag in the increase of cellulase activity, it is suggested that translational as well as transcriptional controls are exerted on the biosynthesis of cellulase in vivo
Summary
Materials--(3H]poly(IJ), K+ salt (specific activity 47 Ci per mole of phosphorus), was from Schwarz-Mann; L-[U-“C]leucine (specific activity 300 mCi per mmol), I,-[4,S3H]leucine (specific activity 39 Ci per mmol), and [2-3H]glycine (specific activity 9.4Ci per mmol) were from New England Nuclear.Creatine phosphate kinase (Li salt) was from Boehringer-MannheimBiochemicals. Materials--(3H]poly(IJ), K+ salt (specific activity 47 Ci per mole of phosphorus), was from Schwarz-Mann; L-[U-“C]leucine (specific activity 300 mCi per mmol), I,-[4,S3H]leucine (specific activity 39 Ci per mmol), and [2-3H]glycine Ci per mmol) were from New England Nuclear. Creatine phosphate kinase (Li salt) was from Boehringer-Mannheim. Research Inc., Waltham, Mass., and tobacco mosaic virus RNA was a gift from. Growth and Treatment of Pla?~ls-Pisum sat&m, L., var. Alaska seeds were grown in darkness for 7 to 8 days and intact seedlings were treated with (2,4-dichlorophenoxy)acetic acid as described in the preceding paper [6]. Subapical segments (10 mm) were excised and used as source of cellulase and polysomes
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