Chatterjee N K, Tuchowski C, Eagan G E, Haley T M
Biochem J. 1984 Mar 1;218(2):583-90. doi: 10.1042/bj2180583.
RNA molecules from nuclear and cytoplasmic polyribosomes of adenovirus-infected HeLa cells were compared by hybridization to analyse the sequence content. Nuclear polyribosomes were released by exposure of intact detergent-washed nuclei to poly(U) and purified. Cytoplasmic polyribosomes were also purified from the same cells. To show that nuclear polyribosomes contain ribosomes linked by mRNA, polyribosomes were labelled with methionine and uridine in the presence of actinomycin D in adenovirus-infected cells. Purified nuclear polyribosomes were treated with EDTA under conditions which dissociate polyribosomes into ribosomes and subunits with a simultaneous release of mRNA, and sedimented. The treatment dissociated these polyribosomes, releasing the mRNA from them. Radiolabelled total RNA from each polyribosome population was fractionated in sucrose gradients into several pools or hybridized to intact adenovirus DNA to select virus-specific RNA. Sucrose-gradient-fractionated pool-3 RNA (about 28S) and virus-specific RNA were then hybridized to fragments of adenovirus DNA cleaved by restriction endonucleases SmaI, HindIII and EcoRI by the Southern-blot technique and by filter hybridization. The results showed that nuclear RNA contained sequences, from about 0 to 18 map units, which were essentially absent from cytoplasmic RNA. Furthermore, the amount of virus-specific RNA for a particular sequence was also different in the two populations.
通过杂交比较腺病毒感染的HeLa细胞核和细胞质多核糖体中的RNA分子，以分析其序列组成。完整的经去污剂洗涤的细胞核经聚（U）处理后释放出核多核糖体并进行纯化。细胞质多核糖体也从相同细胞中纯化得到。为了表明核多核糖体含有通过mRNA连接的核糖体，在腺病毒感染的细胞中，多核糖体在放线菌素D存在下用甲硫氨酸和尿苷进行标记。纯化的核多核糖体在能使多核糖体解离为核糖体和亚基并同时释放mRNA的条件下用EDTA处理，然后进行沉降。该处理使这些多核糖体解离，从中释放出mRNA。来自每个多核糖体群体的放射性标记总RNA在蔗糖梯度中分级分离成几个组分，或与完整的腺病毒DNA杂交以选择病毒特异性RNA。然后，通过Southern印迹技术和滤膜杂交，将蔗糖梯度分级分离的组分3 RNA（约28S）和病毒特异性RNA与经限制性内切酶SmaI、HindIII和EcoRI切割的腺病毒DNA片段杂交。结果表明，核RNA含有约0至18个图谱单位的序列，而这些序列在细胞质RNA中基本不存在。此外，两个群体中特定序列的病毒特异性RNA量也不同。
