Abstract
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.
Highlights
Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts
The mRNA 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 polyacrylamide gels; it sediments as a single peak in sucrose gradients containing 70% formamide
When 12SI-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, the mRNA appears to be approximately
Summary
White Leghorn laving hens were purchased from Rich-G10 Farm, LaGrange, Texas; the&’ oviducts w&e rinsed with cold 0.9% NaCl solution (saline) and frozen in liquid nitrogen immediately after dissection. Liouified nhenol was purchased from Fisher Scientific Co., New Jersey, dnd redistilled before use. G-25 were purchased from Pharmacia Fine Chemicals, Sweden. Creatine phosphokinase, and sodium dodecyl sulfate were from Sigma Chemical Co. Wheat germ was kindly supplied as a gift by General Mills, Inc., Minneapolis, Minn. [‘Clvaline (260 mCi/mmol), ultrapure urea, and sucrose (ribonuclease-free) were purchased from Schwarz/Mann, Orangeburg, N. Ribonuclease T, was obtained from Sankyo Co., Ltd., Japan, and pancreatic ribonuclease A was from Worthington. Poly(A) standards of various chain lengths as determined bv sedimentation velocitv studies were obtained from Miles
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