Quantitative Measurement of Ovalbumin Messenger Ribonucleic Acid Activity

Abstract

Abstract Procedures are described for the quantitative assay of ovalbumin mRNA in a rabbit reticulocyte lysate protein-synthesizing system. A technique for rapid and efficient separation of labeled immunoprecipitated ovalbumin from nonspecific radioactivity is presented. The capacity of the lysate system for total de novo protein synthesis, determined with the use of aurintricarboxylic acid, was found to be correlated with the capacity for ovalbumin mRNA translation. Hemin, a compound that stimulates hemoglobin synthesis in reticulocyte lysates, also stimulates ovalbumin synthesis. The yield of ovalbumin mRNA activity was used as a criterion in choosing optimal conditions for the isolation of undegraded nucleic acid from oviduct. Phenol extraction of total oviduct homogenate yielded only one-third as much activity as isolation by sodium dodecyl sulfate sucrose gradient. When high concentrations of heparin were used to inhibit ribonuclease, the degradation rate of ovalbumin mRNA activity after homogenization could be slowed enough (half-life = 6.4 hours) to permit nearly quantitative recovery of activity in subcellular fractions. No translation of mRNA fragments could be detected in partially degraded nucleic acid preparations. Seventy-one per cent of the total ovalbumin mRNA activity sedimented with polysomes with only 5% in the 27,000 x g pellet. Administration of estrogen to immature chicks induced ovalbumin mRNA activity, and continued hormone treatment was necessary for the maintenance of activity. In vivo administration of actinomycin D to chicks led to a 65% increase in the rate of ovalbumin synthesis in culture with no change in total or polysomal mRNA levels, providing direct evidence that this drug increases the rate of ovalbumin mRNA translation.