【摘要】 从人工贫血的北京鸭网织红细胞中直接提取总RNA,经Oligo(dT)-纤维素柱层析分离获得珠蛋白mRNA,并经蔗糖密度梯度离心首次得到了电泳单一条带的北京鸭球蛋白mRNA。从凝胶电泳以及蔗糖密度梯度离心鉴定其沉降系数为9S。在麦胚无细胞体外翻译体系中测定了它们的蛋白翻译活力。鸭珠蛋白mRNA促进了~3H-亮氨酸参入新生蛋白的活力,达到对照组的10倍。所翻译的蛋白产物在SDS-聚丙烯酰胺凝胶上的电泳行为与天然鸭珠蛋白一致。 经Oligo(dT)-纤维素及蔗糖密度梯度离心提纯的珠蛋白mRNA,在AMV反转录酶及DNA聚合酶的作用下,分别合成了单链及双链cDNA。其双链链长,经凝胶电泳分析,约为500碱基对。更多还原

【Abstract】 Peking duck globin mRNA was purified from the total RNAs of reticulocytes by oligo (dT) -cellulose chromatography and sucrose gradient centrifugation. The purified 9S mRNA showed a single band on a polyacrylamide gel in 7mol/L urea. Their translation activities were measured in a wheat germ cell-free system. In comparison with the control, globin mRNA gave a 10-fold stimulation of 3H-leucine incorporation and the translated products were identified by sodium dodecyl sulfate gel electrophoresis as the authentic globin. Single and double stranded globin cDNA was synthesized from the purified 9S mRNA or reticulocyte poly(A) RNA with AMY reverse transcriptase and DNA polymerase I(Klenow fragment). The products were analyzed by polyacrylamide gel electrophoresis in 7mol/L urea. The lengths of the double stranded cDNA fragments were about 500 base pairs.更多还原