Identification and Isolation of Ovalbumin-synthesizing Polysomes

作者：Richard D. Palmiter, Rafael Palacios, ROBERT T. SCHIMKE · 发表于：Journal of Biological Chemistry · 年份：1972 · DOI：10.1016/s0021-9258(19)45245-9 · 被引用次数：101 · 研究领域：RNA Research and Splicing、RNA and protein synthesis mechanisms、Viral Infectious Diseases and Gene Expression in Insects

Immunological techniques have been developed for the quantification and isolation of polysomes involved in ovalbumin synthesis. For the quantification of ovalbumin polysomes, two methods have been developed; one is based on the binding of 125I-anti-ovalbumin, the other is based on the immunoprecipitation of polysomes. There is a good correlation between the relative rate of ovalbumin synthesis and the extent of 125I-anti-ovalbumin binding to ribosomes or the amount of RNA extracted from polysome immunoprecipitates. With special care to exclude ribonuclease activity, RNA can be obtained from precipitated polysomes in an undegraded state. Such RNA can direct the synthesis of complete ovalbumin chains in an in vitro protein-synthesizing system from rabbit reticulocytes. RNA extracted from polysomes not precipitated by anti-ovalbumin antibody is incapable of directing ovalbumin synthesis. Recovery of messenger RNA activity is essentially quantitative, and the specific activity of ovalbumin mRNA is increased 2.5-fold over that obtained from total polysomes. Nascent chains obtained from immunoprecipitated polysomes are of the size of ovalbumin and smaller, in contrast to nascent chains from total polysomes which contain larger size classes. The results in total indicate that ovalbumin-synthesizing polysomes can be immunoprecipitated quantitatively in an undegraded state, and that ovalbumin mRNA can be extracted from such immunoprecipitates. Immunoprecipitation of polysomes may pote...