Champier J, Rousset B, Berger M
Mol Cell Biochem. 1983;56(2):123-9. doi: 10.1007/BF00227212.
Bovine thyroid RNA labeled by incubation of slices in the presence of 32P-orthophosphate were fractionated by a two-step procedure. Total RNA were extracted by gel filtration on AcA 22 in the presence of pronase and separated by Sepharose 2B chromatography. A small fraction of heavily-labeled RNA (giant RNA) was obtained in the void volume (peak I); the major fraction of RNA (smaller than 45 S) was retarded on the column (peak II) and had a low specific radioactivity. Labeled and total RNA of peak I and labeled RNA species of peak II had a DNA-like nucleotide composition and were polyadenylated. In contrast, the nucleotide composition of total RNA of peak II was similar to that of ribosomal RNA and had a very low poly (adenylic acid) content. Pulse-chase experiments showed a precursor-product relationship between the two RNA fractions. These data indicate that labeled RNA of peak I and peak II likely correspond to newly-synthetized pre-mRNA and mRNA, respectively. Thyrotropin induced a decrease in the amount of 32P-labeled pre-mRNA and a proportional increase of 32P-labeled mRNA suggesting a stimulatory effect of the hormone on the degradation of pre-mRNA.
通过在32P-正磷酸盐存在下孵育切片标记的牛甲状腺RNA,采用两步法进行分级分离。在链霉蛋白酶存在下,通过在AcA 22上进行凝胶过滤提取总RNA,并通过琼脂糖2B色谱法进行分离。在空体积(峰I)中获得一小部分高标记RNA(巨型RNA);大部分RNA(小于45 S)在柱上被阻滞(峰II),且比放射性较低。峰I的标记RNA和总RNA以及峰II的标记RNA种类具有类似DNA的核苷酸组成并且被聚腺苷酸化。相比之下,峰II的总RNA的核苷酸组成与核糖体RNA相似,且聚(腺苷酸)含量非常低。脉冲追踪实验表明这两个RNA组分之间存在前体-产物关系。这些数据表明,峰I和峰II的标记RNA可能分别对应于新合成的前体mRNA和mRNA。促甲状腺激素导致32P标记的前体mRNA量减少,32P标记的mRNA成比例增加,表明该激素对前体mRNA的降解有刺激作用。