Woo S L, Rosen J M, Liarakos C D, Choi Y C, Busch H, Means A R
J Biol Chem. 1975 Sep 10;250(17):7027-39.
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.
变性条件下的制备性琼脂糖凝胶电泳已成功用于从母鸡输卵管中纯化大量卵清蛋白mRNA。如此制备的mRNA在分析性酸-尿素琼脂糖凝胶和含甲酰胺的中性聚丙烯酰胺凝胶中电泳时作为单一成分迁移，基于此其在物理性质上是均一的；在含70%甲酰胺的蔗糖梯度中它也以单一峰沉降。该mRNA在化学上不含核糖体RNA污染，因为其经完全T1核糖核酸酶消化后的寡核苷酸指纹图谱中不包含核糖体RNA可检测到的特异性大寡核苷酸标记。它也未被其他生物活性信使RNA污染，因为当将其添加到无细胞小麦胚芽翻译系统中时，经十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳和特异性免疫沉淀分析，合成的唯一蛋白质产物是卵清蛋白。卵清蛋白mRNA的核苷酸组成为32.3%A、21.0%G、25.7%U和20.7%C [（A+U）/（G+C）等于1.41]。该mRNA含有一个异质性多聚腺苷酸尾，长度在20至140个残基之间，平均链长为62个腺苷酸残基。在完全变性条件下通过电子显微镜测定，纯化的mRNA钠盐的分子量约为650,000±63,000，对应链长为1890±180个核苷酸。该值与通过以下方法获得的值非常一致：（a）在70%甲酰胺存在下的蔗糖梯度离心；（b）评估mRNA中的多聚腺苷酸含量及其多聚腺苷酸尾的平均链长；以及（c）在3%甲醛存在下的沉降速度研究。当让125I标记的卵清蛋白mRNA与大量过量的鸡DNA杂交时，观察到的杂交动力学表明mRNA与鸡DNA的重复序列之间没有明显反应，尽管该mRNA似乎比编码卵清蛋白所需的长度大约长600个核苷酸。因此，看来整个卵清蛋白mRNA主要是从鸡基因组中的一个独特序列转录而来的。
