Cannistraro V J, Hwang P, Kennell D E
Department of Microbiology and Immunology, Washington University School of Medicine, St. Louis, MO 63110.
J Biochem Biophys Methods. 1987 Jul;14(4):211-21. doi: 10.1016/0165-022x(87)90010-8.
Procedures are described for identification of very infrequent in vivo 3'-ends of RNA. After purification by filter hybridization, the 3'-ends were labeled with [5'-32P] cytosine-3'-P in the RNA ligase reaction. Significantly fewer counts were incorporated in the ligase reaction than in the polynucleotide kinase reaction to label 5'-ends. The incorporation was increased by increasing the RNA concentration 5-10 fold by using only one round of filter hybridization. Non-specific RNA binding could be eliminated by RNase A treatment of the filter if a great excess of denatured heterologous DNA was immobilized along with the DNA probe. Significant amounts of DNA were released when eluting the hybrid RNA from such filters. DNA inhibited the ligase reaction, while its DNase products were even more inhibitory. Treatment of the DNase products with alkaline phosphatase completely eliminated the inhibition. We detected no spurious 5'- or 3'-ends generated in the hybrid RNA by RNase A activity used to reduce the non-specific RNA. Also, RNase T1 could be used in place of RNase A to eliminate non-specific RNA binding, but about 25 times more RNase T1 (microgram/microgram) was needed. We used partial alkali digestion to sequence 3'-ends. A major (one hit) and minor (two hit) set of products were produced which could be distinguished from each other by alkaline phosphatase treatment and homochromatography of the products.
本文描述了鉴定RNA体内极少见的3'末端的方法。通过滤膜杂交纯化后，在RNA连接酶反应中用[5'-32P]胞嘧啶-3'-P标记3'末端。与用于标记5'末端的多核苷酸激酶反应相比，连接酶反应中掺入的放射性计数明显较少。通过仅进行一轮滤膜杂交将RNA浓度提高5至10倍，掺入量增加。如果在固定DNA探针的同时固定大量变性的异源DNA，用RNase A处理滤膜可消除非特异性RNA结合。从此类滤膜上洗脱杂交RNA时会释放出大量DNA。DNA抑制连接酶反应，而其DNase产物的抑制作用更强。用碱性磷酸酶处理DNase产物可完全消除抑制作用。我们未检测到用于减少非特异性RNA的RNase A活性在杂交RNA中产生的假5'或3'末端。此外，可用RNase T1代替RNase A来消除非特异性RNA结合，但所需的RNase T1量（微克/微克）约为RNase A的25倍。我们用部分碱消化法对3'末端进行测序。产生了一组主要产物（一次击中）和一组次要产物（两次击中），通过碱性磷酸酶处理和产物的同系层析可将它们区分开来。
