Physical and chemical characterization of purified ovalbumin messenger RNA

Journal of Biological Chemistry (Impact Factor: 4.57). 10/1975; 250(17):7027-39.
Source: PubMed


Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.

Download full-text


Available from: Anthony R Means,
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cellulose-bound DNA complementary to ovalbumin mRNA was used in a continous hybridization system to isolate single-stranded DNA molecules containing the ovalbumin gene. Fragmented DNA segments containing the ovalbumin gene were enriched 300-350 fold in one cycle of purification. Two cycles of purification resulted in a DNA fraction which was enriched 2300 fold in the ovalbumin sequence. The method is suitable for purification of the ovalbumin sequence from both sheared DNA fragments, as well as larger molecular weight DNA containing more than twice the number of nucleotides necessary to code for ovalbumin mRNA. The chromatographic procedures were specific and reproducible. In addition, the recovery of ovalbumin DNA was essentially quantitative (80-100%), even when large amounts of starting DNA (70-75 mg) were used. This purification scheme should also be useful for the enrichment of other unique sequence gened form eucaryotic DNA.
    Cell 04/1976; 7(3):331-8. DOI:10.1016/0092-8674(76)90162-8 · 32.24 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We present evidence that the poly(A) sequence at the 3' end of ovalbumin mRNA has an effect on its translational efficiency in a reticulocyte lysate cell-free system. Polynucleotide phosphorylase has been used to remove selectively the poly(A) while leaving the rest of the molecule intact. It is shown that the stability of the mRNA in a cell free system is not appreciably affected by this procedure. Measurements of the size of ovalbumin-synthesizing polysomes, rate of peptide elongation, and number of rounds of translation per messenger show a generally reduced efficiency for deadenylated mRNA compared to native mRNA. No comparable difference was observed in experiments with a wheat germ cell-free system, which gives few rounds of translation per mRNA. This indicates that the effect results from a lowering of the efficiency of reinitiation on deadenylated mRNA.
    Cell 06/1976; 8(1):51-8. DOI:10.1016/0092-8674(76)90184-7 · 32.24 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The incorporation of [35S]methionine into ovalbumin, a protein containing NH2-terminal N-acetylglycine, has been studied in chicken oviduct magnum cells. The purification of [35S]methionine-labeled ovalbumin from total oviduct proteins was accomplished by dialysis of a crude extract at pH 3.6 followed by chromatography on carboxymethyl cellulose. The radioactive ovalbumin eluted from the column in three peaks (P0, P1, and P2-containing 0, 1, and 2 moles of phosphate, respectively, per mole of ovalbumin). The kinetics of labeling of peaks P0 and P1 showed that the ratio of radioactivity in NH2-terminal methionine to total incorporation was greater at 2 min of labeling than at later times. The transient labeling of the NH2-terminus of ovalbumin with methionine indicates that methionine is the initiator amino acid for the synthesis of this protein, which in its mature form contains NH2-terminal N-acetylglycine.
    Archives of Biochemistry and Biophysics 09/1976; 175(2):730-6. DOI:10.1016/0003-9861(76)90566-X · 3.02 Impact Factor
Show more