Chromatography of ovalbumin messenger ribonucleic acid on complementary deoxyribonucleic acid-cellulose.
DNA complementary to ovalbumin mRNA and covalently bound to cellulose (cDNA-cellulose) was synthesized using avian myeloblastosis virus RNA-directed DNA polymerase. High concentrations of actinomycin D (200 migrogram/ml) were required to produce 97% inhibition of double-stranded DNA synthesis, but mRNA transcription was only slightly inhibited (14%). The conditions used for binding of mRNA to cDNA-cellulose permitted complete hybridization of ovalbumin mRNA in 10 min while stable poly(A):(dT) hybrids failed to form. The temperature at which 50% of the ovalbumin mRNA activity was eluted from cDNA-cellulose was 62 degrees in 0.01 M Tris.HCl. When a batchwise procedure of hybridization and elution was used, the total recovery of ovalbumin mRNA activity applied to the cDNA-cellulose was greater than 98%, indicating little if any degradation of mRNA. Ovalbumin mRNA activity eluted in each chromatographic run was 50 to 70% of that originally used for the synthesis of the cDNA-cellulose. When total polysomal RNA was subjected to chromatography, the bound fraction consisted of ovalbumin mRNA, rRNA, and material behaving like fragments of ovalbumin mRNA. Applying this fraction to cDNA-cellulose a second time eliminated the rRNA but not the presumptive fragments. Ovalbumin mRNA purified either once or twice was enriched between 43- and 56-fold over polysomal RNA in translational activity.
D000818:Animals; D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D002846:Chromatography, Affinity; D004247:DNA / Q000096:biosynthesis; D003609:Dactinomycin / Q000494:pharmacology; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010047:Ovalbumin / Q000096:biosynthesis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism
D012333:RNA, Messenger; D003609:Dactinomycin; D010047:Ovalbumin; D004247:DNA; D012194:RNA-Directed DNA Polymerase