POLY(ADENYLIC ACID)-CONTAINING AND POLY(ADENYLIC ACID)-DEFICIENT MESSENGER-RNA OF MOUSE-LIVER
POLY(ADENYLIC ACID)-CONTAINING AND POLY(ADENYLIC ACID)-DEFICIENT MESSENGER-RNA OF MOUSE-LIVER
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DOI：
10.1016/0005-2787(81)90221-5
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
DOYLE, D
中科院分区：
文献类型：
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作者：
MOFFETT, RB;DOYLE, D
用寡核苷酸(DT)层析分离RNA，并将其分成含聚(A)和缺失聚(A)两类。约99%的聚(A)材料结合到寡聚(DT)上；未结合的材料含有相当短的聚(A)链。所有的RNA组分都保留了启动无细胞翻译的能力，其中缺乏Poly(A)的部分包含了总翻译活性的一半，即mRNA。对无细胞翻译产物的双向聚丙烯酰胺凝胶分析显示，有3种类型的信使核糖核酸：优先含有聚(A)的信使核糖核酸，包括丰富的肝脏信使核糖核酸；缺乏多聚(A)的信使核糖核酸，包括许多中等和低丰度的信使核糖核酸，在含多聚(A)的组分中污染小于10%；以及双晶型的信使核糖核酸介于含聚(A)和缺陷组分之间。C[互补]DNA杂交进一步鉴定了含Poly(A)和双晶型的mRNA类别。测定了不同RNA组分对合成cDNA量的影响。与总RNA相比，含聚(A)的RNA保留了70%的启动能力，而缺失聚(A)的部分则保留了20%的启动能力。将含Poly(A)的、缺Poly(A)的和总的RNA组分与由(+)Poly(A)RNA合成的cDNA杂交。含有Poly(A)的RNA与平均R0t1/2[R0=RNA浓度；t1/2=杂交的一半时间]杂交的速度大约是总RNA的20倍。Poly(A)缺失的RNA与平均R0t1/2杂交的速度大约是总RNA的3-4倍。这些R0t1/2移位表明，在含聚(A)的组分中回收了超过3/4的可杂交RNA，而在缺乏聚(A)的RNA组分中回收的不到1/4。在异源杂交中，丰度级别不太明显。在所有的情况下，杂交的程度是相似的，这表明虽然不同的RNA组分中不同的RNA物种的数量不同，但大多数杂交物种的RNA存在于每个RNA组分中。纯化了丰富类mRNA的cDNA，并与(+)-和(-)Poly(A)RNA杂交。与丰度较高的物种相对应的信使RNA在含聚(A)的组分中的丰度至少是丰度较低的信使RNA的2倍，只有不到10%的丰度的信使RNA出现在聚(A)缺乏的组分中。
RNA was isolated and fractionated into poly(A)-containing and -deficient classes by oligo(dT) chromatography. Approximately 99% of the poly(A) material bound to the oligo(dT); that which did not bind contained substantially shorter poly(A) chains. All RNA fractions retained an ability to initiate cell-free translation, with the poly(A)-deficient fraction containing half the total translational activity, i.e., mRNA. Two-dimensional polyacrylamide gel analysis of the cell-free translation products revealed 3 classes of mRNA: mRNA preferentially containing poly(A), including the abundant liver mRNA species; poly(A)-deficient mRNA, including many mid- and low-abundant mRNA exhibiting less than 10% contamination in the poly(A)-containing fraction; and bimorphic species of mRNA proportioned between both the poly(A)-containing and -deficient fractions. Poly(A)-containing and bimorphic mRNA classes were further characterized by c[complementary]DNA hybridizations. The capacity of various RNA fractions to prime cDNA synthesis was determined. Compared to total RNA, the poly(A)-containing RNA retained 70% of the priming capacity, while 20% was found in the poly(A)-deficient fraction. Poly(A)-containing, poly(A)-deficient and total RNA fractions were hybridized to cDNA synthesized from (+)poly(A)RNA. Poly(A)-containing RNA hybridized with an average R0t1/2 [R0 = concentration of RNA; t1/2 = half-time of hybridization] approximately 20 times faster than total RNA. Poly(A)-deficient RNA hybridized with an average R0t1/2 approximately 3-4 times slower than total RNA. These R0t1/2 shifts indicated that in excess of 3/4 of the total hybridizable RNA was recovered in the poly(A)-containing fraction and that less than 1/4 was recovered in the poly(A)-deficient RNA fraction. Abundancy classes were less distinct in heterologous hybridizations. In all cases the extent of hybridization was similar, indicating that while the amount of various mRNA species varied among the RNA fractions, most hybridizing species of RNA were present in each RNA fraction. cDNA to the abundant class of mRNA was purified and hybridized to both (+)- and (-)poly(A)RNA. Messenger RNA corresponding to the more abundant species was enriched in the poly(A)-containing fraction at least 2-fold over the less abundant species of mRNA, with less than 10% of the abundant mRNA appearing in the poly(A)-deficient fraction.