Preparation and preliminary characterization of purified ovalbumin messenger RNA from the hen oviduct - PubMed (original) (raw)
Preparation and preliminary characterization of purified ovalbumin messenger RNA from the hen oviduct
J M Rosen et al. Biochemistry. 1975.
Abstract
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.
Similar articles
- Physical and chemical characterization of purified ovalbumin messenger RNA.
Woo SL, Rosen JM, Liarakos CD, Choi YC, Busch H, Means AR, O'Malley. Woo SL, et al. J Biol Chem. 1975 Sep 10;250(17):7027-39. J Biol Chem. 1975. PMID: 1158896 - Partial purification of a progesterone-inducible messenger RNA (avidin) from hen oviduct.
Sperry PJ, Woo SL, Means AR, O'Malley BW. Sperry PJ, et al. Endocrinology. 1976 Jul;99(1):315-25. doi: 10.1210/endo-99-1-315. Endocrinology. 1976. PMID: 939199 - Isolation and characterization of purified rat casein messenger ribonucleic acids.
Rosen JM. Rosen JM. Biochemistry. 1976 Nov 30;15(24):5263-71. doi: 10.1021/bi00669a011. Biochemistry. 1976. PMID: 999806 - Immunochemical isolation and characterization of ovalbumin messenger ribonucleic acid.
Shapiro DJ, Schimke RT. Shapiro DJ, et al. J Biol Chem. 1975 Mar 10;250(5):1759-64. J Biol Chem. 1975. PMID: 1112829 - A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.
Gedamu L, Iatrou K, Dixon GH. Gedamu L, et al. Biochem J. 1978 Jun 1;171(3):589-99. doi: 10.1042/bj1710589. Biochem J. 1978. PMID: 666728 Free PMC article.
Cited by
- Ending the message: poly(A) signals then and now.
Proudfoot NJ. Proudfoot NJ. Genes Dev. 2011 Sep 1;25(17):1770-82. doi: 10.1101/gad.17268411. Genes Dev. 2011. PMID: 21896654 Free PMC article. Review. - Regulation of casein messenger RNA during the development of the rat mammary gland. 1975.
Rosen JM, Woo SL, Comstock JP. Rosen JM, et al. J Mammary Gland Biol Neoplasia. 2009 Sep;14(3):343-51. doi: 10.1007/s10911-009-9143-7. Epub 2009 Aug 5. J Mammary Gland Biol Neoplasia. 2009. PMID: 19653075 No abstract available. - Mechanisms by which eucaryotic genes evolve.
O'Malley BW. O'Malley BW. Breast Cancer Res Treat. 1981;1(4):327-37. doi: 10.1007/BF01806748. Breast Cancer Res Treat. 1981. PMID: 7348579 - Organization of rRNA structural genes in the archaebacterium Thermoplasma acidophilum.
Tu J, Zillig W. Tu J, et al. Nucleic Acids Res. 1982 Nov 25;10(22):7231-45. doi: 10.1093/nar/10.22.7231. Nucleic Acids Res. 1982. PMID: 7155894 Free PMC article. - Blotting of RNA onto ion exchange paper allowing subsequent characterization by in situ translation in addition to blot hybridization.
Saris CJ, Franssen HJ, Heuyerjans JH, van Eenbergen J, Bloemers HP. Saris CJ, et al. Nucleic Acids Res. 1982 Aug 25;10(16):4831-43. doi: 10.1093/nar/10.16.4831. Nucleic Acids Res. 1982. PMID: 7134000 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Other Literature Sources