Biomaterial Database

Preparation and preliminary characterization of purified ovalbumin messenger RNA from the hen oviduct. 1975

J M Rosen, and S L Woo, and J W Holder, and A R Means, and B W O'Malley

Preparation of milligram amounts of purified ovalbumin mRNA was accomplished by a sequential combination of precise sizing techniques with the selective purification of the poly(A) containing RNA by either affinity chromatography or adsorption to nitrocellulose filters. Several new techniques were applied to the purification of ovalbumin 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 preparative sacle 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 containing all of the ovalbumin mRNA activity was also found using linear formamide-sucrose gradients. (2) Determination of both total mRNA activity and ovalbumin mRNA activity in the wheat germ cell-free translation assay revealed that 92% of the total peptides synthesized were specifically immunoprecipitable with an ovalbumin antiserum. (3) Analysis of the total peptides synthesizied in the wheat germ assay by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of a single radioactive 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-containing sucrose gradients yielded a value of 520,000 or approximately 1600 nucleotides. This value was considerably less than the value of 900,000 obtained by gel electrophoresis 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 properties.

UI	MeSH Term	Description	Entries
D010047	Ovalbumin	An albumin obtained from the white of eggs. It is a member of the serpin superfamily.	Serpin B14
D010057	Oviducts	Ducts that serve exclusively for the passage of eggs from the ovaries to the exterior of the body. In non-mammals, they are termed oviducts. In mammals, they are highly specialized and known as FALLOPIAN TUBES.	Oviduct
D002499	Centrifugation, Density Gradient	Separation of particles according to density by employing a gradient of varying densities. At equilibrium each particle settles in the gradient at a point equal to its density. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Centrifugations, Density Gradient,Density Gradient Centrifugation,Density Gradient Centrifugations,Gradient Centrifugation, Density,Gradient Centrifugations, Density
D002645	Chickens	Common name for the species Gallus gallus, the domestic fowl, in the family Phasianidae, order GALLIFORMES. It is descended from the red jungle fowl of SOUTHEAST ASIA.	Gallus gallus,Gallus domesticus,Gallus gallus domesticus,Chicken
D002846	Chromatography, Affinity	A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D005260	Female		Females
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D012270	Ribosomes	Multicomponent ribonucleoprotein structures found in the CYTOPLASM of all cells, and in MITOCHONDRIA, and PLASTIDS. They function in PROTEIN BIOSYNTHESIS via GENETIC TRANSLATION.	Ribosome
D012333	RNA, Messenger	RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.	Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated