Preparation and preliminary characterization of purified ovalbumin messenger RNA from the hen oviduct

Abstract

Preparation of milligram amounts of purified ovalbumin mRNA was accomplished by a sequential com- bination of precise sizing techniques with the selective puri- fication of the poly(A) containing RNA by etiher affinity chromatography or adsorption to nitrocellulose filters. Sev- eral new techniques were applied to the purification of oval- bumin mRNA including Sepharose 4B chromatography and agarose gel electrophoresis in the presence of 6 M urea at pH 3.5. All the procedures used were adapted on a pre- parative scale to the fractionation of large quantities of RNA. The purity of the ovalbumin mRNA was assessed by several independent criteria. (1) Purified ovalbumin mRNA migrated as a single band during both agarose-urea and formamide-polyacrylamide gel electrophoresis at pH 3.5 and 7.4, respectively. A single absorbance peak contain- ing all of the ovalbumin mRNA activity was also found using linear formamide-sucrose gradients. (2) Determina- tion of both total mRNA activity and ovalbumin mRNA activity in the wheat germ cell-free translation assay re- vealed that 92% of the total peptides synthesized were spe- cifically immunoprecipitable with an ovalbumin antiserum. (3) Analysis of the total peptides synthesized in the wheat germ assay by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of a single ra- dioactive peak that corresponded exactly to a specifically immunoprecipitable ovalbumin standard. Thus, based on these observations ovalbumin mRNA appears to be greater than 95% pure. A preliminary estimation of the molecular weight of purified ovalbumin mRNA by formamide-con- taining sucrose gradients yielded a value of 520,000 or ap- proximately 1600 nucleotides. This value was considerably less than the value of 900,000 obtained by gel electrophore- sis under denaturing conditions. Analysis of the poly(A) content, by a hybridization assay with (3H)poly(U) revealed the presence of a poly(A) region containing approximately 70 adenosine residues. Thus, the size of the ovalbumin mRNA is considerably greater than that required to code for a protein of 387 amino acids. The availability of large quantities of purified ovalbumin mRNA should now permit a more thorough analysis of its physical and chemical prop- erties.