Phillips L A, Park J J, Hollis V W
Proc Natl Acad Sci U S A. 1974 Nov;71(11):4366-70. doi: 10.1073/pnas.71.11.4366.
The location of poly(A) sequences in the RNA of mammalian RNA-tumor viruses was determined by enzymatic analyses. The 56-64S viral genomic RNAs, the 20-40S viral subunit RNAs, and the 4-5S poly(A) sequences excised from these viral RNAs were subjected to either hydrolysis with a 3'-OH specific exoribonuclease from Ehrlich ascites tumor cells or phosphorolysis from the 3'-termini with polynucleotide phosphorylase from Micrococcus luteus. Purified adenosine-labeled poly(A) fragments, excised from genomic viral RNAs by RNase A and T(1) digestion, were hydrolyzed with the 3'-OH specific exoribonuclease for various periods of time. Poly(U) filter binding studies of the residual poly(A) indicated that 97% of the poly(A) fragments were hydrolyzed. Adenosine-labeled genomic and subunit viral RNAs and excised poly(A) fragments were phosphorolyzed from their 3'-termini for various periods of time with polynucleotide phosphorylase. The degree of phosphorolysis was monitored by poly(U) filter binding studies, and CCl(3)COOH insolubility and solubility determinations. There was an initial preferential rate of phosphorolysis of the poly(A) sequences of genomic and subunit viral RNAs as compared to the total adenosine-labeled viral RNAs. The data from these two different enzymatic mechanisms of action indicated conclusively that the poly(A) sequences were located at the 3'-termini of genomic and subunit viral RNAs.
通过酶促分析确定了哺乳动物RNA肿瘤病毒RNA中聚腺苷酸(poly(A))序列的位置。对56 - 64S病毒基因组RNA、20 - 40S病毒亚基RNA以及从这些病毒RNA中切下的4 - 5S聚(A)序列,用艾氏腹水瘤细胞的3'-OH特异性外切核糖核酸酶进行水解,或用藤黄微球菌的多核苷酸磷酸化酶从3'末端进行磷酸解。通过核糖核酸酶A和T(1)消化从基因组病毒RNA中切下的纯化腺苷标记的聚(A)片段,用3'-OH特异性外切核糖核酸酶水解不同时间。对残留聚(A)的聚尿苷酸(poly(U))滤膜结合研究表明,97%的聚(A)片段被水解。用多核苷酸磷酸化酶从腺苷标记的基因组和亚基病毒RNA以及切下的聚(A)片段的3'末端进行不同时间的磷酸解。通过聚(U)滤膜结合研究、三氯乙酸(CCl(3)COOH)不溶性和溶解性测定来监测磷酸解程度。与总的腺苷标记病毒RNA相比,基因组和亚基病毒RNA的聚(A)序列最初有优先的磷酸解速率。来自这两种不同作用机制的酶促数据确凿地表明,聚(A)序列位于基因组和亚基病毒RNA的3'末端。