Silvertien S, Millette R, Jones P, Roizman B
J Virol. 1976 Jun;18(3):977-91. doi: 10.1128/JVI.18.3.977-991.1976.
Fractionation of polyadenylated RNA from cells infected with herpes simplex virus by affinity chromatography on columns of poly (U) immobilized on glass-fiber filters yielded three major classes of RNA-containing poly(A) chains with average lengths of 30, 50, and 155 adenylate residues [poly(A)30, poly(A)50, poly(A)155]. In contrast, nitrocellulose membranes bound predominantly a fraction of RNA containing poly(A)155. The distribution of cytoplasmic RNA in the three classes was found to be independent of the labeling interval, ranging from 10 min to 6 h. Cytoplasmic poly(A) RNA consisted mainly (57 to 68%) of the poly(A)155 class; this was also the major class (68%) of polyadenylated RNA found in polyribosomes. Nuclear poly(A) RNA consisted largely (42 to 50%) of poly(A)30 class, whereas high-molecular-weight nuclear RNA sedimenting at greater than 45S contained almost exclusively the poly(A)30 tracts. Hybridization experiments involving unlabeled RNA and labeled viral DNA demonstrated the presence of viral RNA sequences complementary to approximately 40% of viral DNA in all polyadenylated RNA classes. Inasmuch as unfractionated cytoplasmic RNA arises from approximately 40% of the viral DNA, we conclude that most, if not all, viral RNA species present in the cytoplasm are adenylated. In contrast to these results, only a fraction of poly(A)155 RNA, complementary to 21% of viral DNA, bound to nitrocellulose filters. The selective binding of poly(A)155 sequences to nitrocellulose filters might be related to its secondary structure, since transcripts homologous to 40% of viral DNA bind to nitrocellulose membranes, provided the RNA is denatured prior to filtration. The data suggest that poly(A) tracts arise by at least two separate steps. The first involves the appearance of poly(A)30 tracts in the high-molecular-weight nuclear transcripts. The second involves polyadenylation to ply(A)50 and poly(A)155 RNA classes concomitant with processing and transport to the cytoplasm.
通过在固定于玻璃纤维滤器上的聚（U）柱上进行亲和层析，从感染单纯疱疹病毒的细胞中分离出多聚腺苷酸化RNA，得到了三类主要的含RNA多聚（A）链，其平均长度分别为30、50和155个腺苷酸残基[多聚（A）30、多聚（A）50、多聚（A）155]。相比之下，硝酸纤维素膜主要结合了一部分含多聚（A）155的RNA。发现这三类细胞质RNA的分布与标记时间间隔无关，标记时间间隔从10分钟到6小时不等。细胞质多聚（A）RNA主要（57%至68%）由多聚（A）155类组成；这也是在多核糖体中发现的多聚腺苷酸化RNA的主要类别（68%）。核多聚（A）RNA很大程度上（42%至50%）由多聚（A）30类组成，而沉降系数大于45S的高分子量核RNA几乎只包含多聚（A）30片段。涉及未标记RNA和标记病毒DNA的杂交实验表明，在所有多聚腺苷酸化RNA类别中，都存在与大约40%病毒DNA互补的病毒RNA序列。由于未分级的细胞质RNA来自大约40%的病毒DNA，我们得出结论，细胞质中存在的大多数（如果不是全部）病毒RNA种类都进行了腺苷酸化。与这些结果相反，只有一部分与21%病毒DNA互补的多聚（A）155 RNA能与硝酸纤维素滤器结合。多聚（A）155序列与硝酸纤维素滤器的选择性结合可能与其二级结构有关，因为与40%病毒DNA同源的转录本在过滤前变性的情况下能与硝酸纤维素膜结合。数据表明，多聚（A）片段至少通过两个独立步骤产生。第一步涉及高分子量核转录本中多聚（A）30片段的出现。第二步涉及多聚腺苷酸化形成多聚（A）50和多聚（A）155 RNA类别，同时伴随着加工和转运到细胞质中。
